CN104288779B - PEGization peptides dendrimer targeting drug delivery system of load gemcitabine and preparation method thereof - Google Patents
PEGization peptides dendrimer targeting drug delivery system of load gemcitabine and preparation method thereof Download PDFInfo
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Abstract
The present invention is to solve the anticancer specific problem of dendrimer drug-supplying system, it is provided that a kind of PEGization peptides dendrimer targeting drug delivery system loading gemcitabine and preparation method thereof.It is characterized in that: drug-supplying system is antitumor drug gemcitabine, target function sex factor GFLG and the conjugate of PEGization many peptides dendrimer, functional dendritic macromole is formed by GFLG after being connected with PEGization peptides dendrimer by gemcitabine, while obtaining good biocompatibility, the medicine of Isodose is made to reach obvious antitumor curative effect.
Description
Technical field
The invention belongs to the preparation field of macromolecule targeted nano carrier, be specifically related to load the PEGization peptide of gemcitabine
Class dendrimer targeting drug delivery system and preparation method thereof.
Background technology
Due to the size being accurately controlled, low polydispersity and can the modified surface of multifunction so that dendrimer
(dendrimer) compared with traditional colloid or macromole carrier systems, it is expected to improve the relevant nature of cancer therapy drug, such as medicine
Thing kinetics, therefore it is highly suitable for as carrier to transmit cancer therapy drug.Owing to peptides dendrimer has merged tree-shaped
The feature of both macromole and peptide quasi-molecule, and obtain water solublity, biodegradability, biocompatibility and immunity phase
The advantages such as capacitive, so in nearest following period of time, peptides dendrimer obtains the biggest pass as a kind of pharmaceutical carrier
Note.Although there being great advantage, but it is because quilt when that such controlled drug delivery systems being easy to circulate in vivo
Quickly metabolism is fallen, and its vivo applications is still faced with challenge and limitation greatly.Although it is this by the situation of tachymetabolism
Can avoid by improving the algebraically of dendrimer, but the difficulty in synthesis and the corresponding toxicity produced but are can not
Avoid.
In order to avoid the problems referred to above, Polyethylene Glycol (PEG) segment is introduced the spherical surface of dendrimer, thus prepares
Go out nanoparticle system based on dendrimer.This system due to its molecular weight and size compared to simple tree-shaped greatly
Molecule has a certain degree of increase, thus obtains longer blood circulation time while reducing system toxicity and preferably swell
Tumor tissue enrichment degree.Meanwhile, PEGization is also prevented from the reticuloendothelium early stage removing for dendrimer.Therefore,
The peptides dendrimer of PEGization is the most attractive controlled drug delivery systems of one, such as " Development of
efficient acid cleavable multifunctional prodrugs derived from dendritic
Polyglycerol with a poly (ethylene glycol) shell " (Journal of Controlled
Release. 2011;151:295-301), " Dendrimers of citric acid and poly (ethylene
Glycol) as the new drug-delivery agents " (Biomaterials. 2005;26:1175-83),
“Pharmacokinetics and tumor disposition of PEGylated, methotrexate conjugated
Poly-L-lysine dendrimers " (Molecular Pharmaceutics. 2009;6:1190-204).But, at present
The environment sensitive key of PEGization number tree macromole drug-supplying system is pH response, i.e. utilizes tumor tissues and inside tumor cells
Acidity is controlled release, and owing to the pH value in the middle of tumor tissues also can be to making sensitive bond fission, free drug is inevitable
Meeting spread in the middle of tumor tissues, increase it and overflow tumor tissues, come back to the probability in the middle of blood vessel, thus reduce it and resist
Cancer specificity.
As pharmaceutical carrier, the nano-particle based on dendrimer for treatment of cancer must be selectively by freely
Drug release enters in the middle of tumor tissues or tumor cell, promote with so can collecting big degree antitumous effect and
Reduce side effects of pharmaceutical drugs.Enzyme sensitivity system is a kind of efficient, has selective drug delivery system, and it is by can only be by
The junction point that the enzyme of tumor cell specific secretion is cracked is connected with medicine.Cathepsin B is a kind of swollen at great majority
The lysosomal cysteine protease of overexpression inside oncocyte and neoplastic epithelial cells.GFLG (Gly-Phe-Leu-Gly)
Being a kind of substrate corresponding to cathepsin B, it is applied to multiple polymers as a kind of specific cleavage small peptide
In the middle of medicine.This tetrapeptide array suffers from good stability in the middle of blood plasma and serum, and can endocytosis it
After in the middle of lysosome discharge medicine.
Summary of the invention
The present invention is to solve the number tree anticancer specific problem of macromole drug-supplying system, the invention provides a kind of load
PEGization peptides dendrimer targeting drug delivery system of gemcitabine and preparation method thereof.By GFLG by gemcitabine and PEG
Change after peptides dendrimer is connected and form functional dendritic macromole, while obtaining good biocompatibility, make same
Isodose medicine reaches obvious antitumor curative effect.
Delivery system based on GFLG system is to rupture for the distinctive enzyme of tumor cell, i.e. delivery system must enter
Specific reaction just occurs after entering tumor cell, and therefore its anticancer specificity is effectively ensured.
For achieving the above object, the present invention adopts the following technical scheme that:
The PEGization peptides dendrimer targeting drug delivery system of load gemcitabine, it is characterised in that: drug-supplying system is anti-
Tumour medicine gemcitabine, target function sex factor GFLG and the conjugate of PEGization peptides dendrimer, its structure is as follows:
Wherein, the structure of A is:
B is 3 ~ 5 generation peptides dendrimers of PEGization, and the terminal amino group of dendrimer is connected with group A.
Dendrimer must rely on bigger size just can embody EPR effect, and (the enhancing infiltration of tumor cell is detained
Effect) and avoid by too early metabolism.Although the dendrimer of high algebraically has a bigger size, but the thing followed
It it is higher system toxicity.
The end of peptides dendrimer of the present invention connects altogether 1 or multiple described A group.
Peptides dendrimer of the present invention is the peptides dendrimer of lysine or glutamic acid.
The grain diameter of PEGization peptides dendrimer targeting drug delivery system of the present invention is 80 ~ 130 nm.This particle diameter
Can effectively utilize the feature that tumor blood vessels wall is loose, by EPR effect, reach the drug-supplying system Gao Fu at tumor tissues
Collection;Can effectively extend again its circulation time in vivo simultaneously, thus be expected to show good antitumous effect.
The carrier of drug-supplying system of the present invention is peptides dendrimer, has monodispersity, and functional group can be modified relatively in periphery
Many, the structure of albuminoid, its catabolite in vivo is natural amino acid, and biological safety is high；The introducing of PEG chain segment is permissible
Improve the water solublity of whole system, the dendrimer of relatively low algebraically can be helped to obtain on the premise of not improving algebraically simultaneously
Bigger nano-scale, improves the biological safety of whole system, and due to the outside modification of PEG so that delivery system is not
Easily caught by reticuloendothelial cell, on this basis, due to the introducing of PEG, make system have hydrophilic region, it is possible to use
Hydrophobe effect is self-assembly of nano-particle；It is sensitive that the introducing of GFLG makes material have the enzyme-specific for tumor cell
Characteristic so that it is can be relatively stable before entering tumor cell, and occur specific enzyme hydrolysis anti-after entering cell
Should, play specific antitumaous effect.
The toxicity of gemcitabine is less, is the first-line drug of the most a lot of cancer chemotherapy, with amido in its molecular structure,
Amido link can be formed so that it is discharge former medicine when fracture with the carboxyl of GFLG end.It addition, the molecular volume of gemcitabine is relatively
Little, by sterically hindered affect little, its In-vitro specificity fracture efficiency significantly improve.
The present invention loads the preparation method of the peptides dendrimer targeting drug delivery system of gemcitabine, it is characterised in that:
On the gemcitabine (GEM) conjugate with GFLG, first introduce methyl ethylene (MA), obtain conjugate MA-GFLG-GEM, standby
With；Again using the peptides dendrimer alkynyl as carrier, dendrimer introduces three benzylthios, obtains alkynyl three
, after sloughing trityl, there is coupling reaction with conjugate MA-GFLG-GEM, obtain alkynyl in benzylthio dendrimer
The conjugate of dendrimer and gemcitabine, after through PEGization finished product.
The present invention introduces MA(methyl ethylene on the gemcitabine conjugate with GFLG), and draw on dendrimer
Entering three benzylthios, trityl (Trt) is as the protection group of sulfydryl, and after sloughing Trt, the double bond of MA is anti-with sulfydryl generation coupling
Should, obtain the conjugate of alkynyl dendrimer and gemcitabine.
Meanwhile, use alkynyl dendrimer, form amido link by the condensation reaction of amino with carboxyl, so that tree
Alkynyl in the peripheral band of shape macromole, provides the reaction site of click-reaction for PEGization below, for the alkynes-nitrine of copper catalysis
Click on cycloaddition (CuAAC) reaction, effectively reduce unnecessary side reaction and keep the low polydispersity of dendrimer.
The preparation method concrete operation step of the PEGization peptides macromole of the present invention is as follows:
The preparation of A MA-GFLG-GEM conjugate
With Boc-GFLG-OMe as raw material, with ethylene methacrylic acyl chloride reaction prepare MA-GFLG-OH, rear MA-GFLG-OH with
DIPEA, tetrahydrothiazole-2-thion react, after reaction terminates, by product, GEMCITABINE HYDROCHLORIDE, pyrrole
Pyridine is dissolved in anhydrous dimethyl sulfoxide, and lucifuge is reacted, and reaction products therefrom is MA-GFLG-GEM conjugate；
The synthesis of B alkynyl dendrimer
With the dendrimer of Boc Yu Cbz radical protection as raw material, reacting with Pb/C under an atmosphere of hydrogen, reaction terminates
After, product is dissolved in the middle of DMF, system adds DIPEA, hexynic acid, BTA-N, N,
N', N'-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole, react under nitrogen protection, and reaction products therefrom is alkynyl
Dendrimer；
This step forms amido link by the condensation reaction of amino with carboxyl, sloughs Cbz, so that outside dendrimer
Alkynyl on shroud, provides the reaction site of click-reaction for PEGization below.
The synthesis of C alkynyl three benzylthio dendrimer
Alkynyl dendrimer is dissolved in the mixed solvent of anhydrous methylene chloride and trifluoroacetic acid, and system is protected at nitrogen
Protect lower reaction, product is dissolved in dry DMF, system adds DIPEA, 3-tri-benzylthio third
Acid, BTA-N, N, N', N'-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole, react, instead under nitrogen protection
Answering products therefrom is alkynyl three benzylthio dendrimer；
The purpose introducing three benzylthios is to introduce sulfydryl in dendrimer periphery, and trityl (Trt) is conduct
The protection group of sulfydryl and exist.Introduce sulfydryl purpose be can be with MA(methyl ethylene on gemcitabine conjugate) double bond
There is mercapto-alkene reaction.
The synthesis of D alkynyl dendrimer-gemcitabine conjugate
Alkynyl three benzylthio dendrimer is dissolved in the mixed solvent of anhydrous methylene chloride and trifluoroacetic acid, system
React under nitrogen protection；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, by product
Be dissolved in the middle of dry DMF, in solution add conjugate MA-GFLG-GEM, 1,8-diazabicylo [5.4.0] 11 carbon-7-
Alkene, under nitrogen protection lucifuge reaction, reaction products therefrom is alkynyl dendrimer-gemcitabine conjugate；
Because sulfydryl-vinyl coupling reaction inherently highly effective reaction being similar to click-reaction, tie at some
Even quantitative response in structure.Rely on this response type, Ah mould can be changed by the change of simple reaction rate of charge
Element is for the degree of modification of alkynyl dendrimer, thus changes the drug loading of final material.
Use TFA(trifluoroacetic acid) purpose be remove mercapto-protective agent: trityl, make sulfydryl come out.At DBU
Effect under, the double bond of MA and sulfydryl generation coupling reaction in MA-GFLG-GEM.
The purpose adding triethyl silicane is the cation of generation in the middle of catching reaction process, promotes the carrying out of reaction,
Could add after must waiting until to become yellow green, otherwise can not play the effect of deprotection agent.
E PEGization dendrimer
In a nitrogen atmosphere, by alkynyl dendrimer-gemcitabine conjugate, CuSO4·5H2O、N3-mPEG, anti-
Bad hematic acid sodium joins DMF and H2In the middle of the mixed solvent of O, reaction system lucifuge is reacted, after reaction terminates, product is purified,
Dialysis, lyophilizing obtain PEGization dendrimer-gemcitabine conjugate.
Preferably, described B and step C, system reacts after terminating under nitrogen protection, and solution with ethyl acetate dilutes,
And use NaHCO successively3、HCl、NaHCO3, NaCl washing, the anhydrous MgSO of organic facies4It is dried, and solvent is distilled off with decompression,
Residue, through recrystallization, obtains product.
The method utilizing cyclic washing and extraction can effectively go the removal of impurity and reduce purification step, beneficially synthetic quantity
Amplify.
Preferably, described D step, in solution add conjugate MA-GFLG-GEM, 1,8-diazabicylo [5.4.0]
After 11 carbon-7-alkene, under nitrogen protection and room temperature, lucifuge is reacted 0.1 ~ 90 hour.It is effectively improved sulfydryl-vinyl coupling anti-
The reaction probability answered.
Preferably, described D step, after reaction terminates, reactant liquor is added dropwise in the middle of ethyl acetate, and precipitate passes through
It is centrifuged, purifies, is dried, obtain alkynyl dendrimer-gemcitabine conjugate.
Can effectively remove unreacted dendrimer and DBU by being added dropwise to ethyl acetate, go the removal of impurity, effectively
Improve product purity.
The beneficial effects of the present invention is:
1, the peptides dendrimer targeting drug delivery system of the present invention, PEG chain segment and GFLG small peptide combine, and can send out
Wave its respective advantage, be the peptides dendrimer system that first GFLG introduced simultaneously, conventional reproducibility can be avoided sensitive
Not only rupture in tumor cell with pH sensitivity key, fracture the most also can be occurred in tumor tissues to discharge medicine
Problem, thus lower medicine and overflow the risk of tumor locus.
2, the grain diameter of the PEGization peptides dendrimer targeting drug delivery system of the present invention can reach 80 ~ 130nm.Should
Particle diameter can effectively utilize the feature that tumor blood vessels wall is loose, by EPR effect, reaches the drug-supplying system height at tumor tissues
Enrichment;Can effectively extend again its circulation time in vivo simultaneously, thus be expected to show good antitumous effect.
3, the preparation method of targeting drug delivery system of the present invention uses and introduces methyl second on gemcitabine and the conjugate of GFLG
Thiazolinyl, then with the scheme of dendrimer coupling, reaction efficiently, preparation method gentle simple, and copper ion can be avoided as far as possible
Introducing.
4, the preparation method of the present invention uses alkynyl dendrimer, formed by the condensation reaction of amino with carboxyl
Amido link, so that alkynyl in the peripheral band of dendrimer, provides the reaction site of click-reaction for PEGization below, for
Alkynes-the nitrine of copper catalysis clicks on cycloaddition (CuAAC) reaction, effectively reduces unnecessary side reaction and keeps tree-shaped big point
The low polydispersity of son.
5, the preparation method of the present invention introduces three benzylthios, its object is to introduce sulfydryl in dendrimer periphery,
Trityl is to exist as the protection group of sulfydryl.The purpose introducing sulfydryl is can be with MA(first on gemcitabine conjugate
Base vinyl) double bond generation mercapto-alkene reaction.
6, the present invention adds triethyl silicane in the dendrimer coupling reaction with gemcitabine, its object is to catch
Catch the cation produced in the middle of course of reaction, promote the carrying out of reaction, and must wait until that solution becomes ability after yellow green
Add, otherwise can not play the effect of removing trityl as protecting group agent.
7, the present invention terminates in the synthetic reaction of alkynyl dendrimer and alkynyl three benzylthio dendrimer
After, solution with ethyl acetate dilutes, and uses NaHCO successively3、HCl、NaHCO3, NaCl washing, utilize cyclic washing and extraction
Method can effectively be gone the removal of impurity and reduce purification step, beneficially the amplification of synthetic quantity.
8, the present invention is after the coupling reaction of dendrimer Yu gemcitabine terminates, by being added dropwise to by reactant liquor
Ethyl acetate can effectively remove unreacted dendrimer and DBU, goes the removal of impurity, is effectively improved product purity.
Accompanying drawing explanation
Fig. 1 is the gemcitabine content standard curve of the couplet I that HPLC measures.
Fig. 2 is the DLS figure of PEGization dendrimer-gemcitabine conjugate.
Fig. 3 is the TEM figure of PEGization dendrimer-gemcitabine conjugate.
Fig. 4 is PEGization dendrimer-gemcitabine couplet and gemcitabine extracorporeal anti-tumor design sketch.
Fig. 5 is that PEGization dendrimer-gemcitabine couplet is thin with the tumor that gemcitabine vitro cytotoxicity is tested
Born of the same parents' survival rate curve chart.
Fig. 6 is non-medicine carrying carrier cytotoxicity Normocellular for the COS-7 figure of PEGization dendrimer.
Fig. 7 is the tumor growth curve figure of tumor-bearing mice.
Fig. 8 is the tumor weight schematic diagram of tumor-bearing mice.
Fig. 9 is the schematic diagram of the tumor control rate of tumor-bearing mice.
Figure 10 is the structural representation of the dendrimer conjugate in the embodiment of the present invention 1.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the essentiality content of the present invention is described in further detail.
Embodiment 1
The PEGization peptides dendrimer targeting drug delivery system of load gemcitabine, drug-supplying system is that antitumor drug is lucky
His shore, west, target function sex factor GFLG and the conjugate of PEGization many peptides dendrimer, its structure as shown in Figure 10:
Couplet I: n=10, the PEGization of m=3 3 generation lysine dendrimer-gemcitabine conjugate
Couplet II: n=1, the PEGization of m=1 3 generation lysine dendrimer-gemcitabine conjugate
Couplet III: n=10, the PEGization of m=1 3 generation lysine dendrimer-gemcitabine conjugate
Couplet IV: n=1, the PEGization of m=1 4 generation lysine dendrimer-gemcitabine conjugate
Couplet V: n=2, the PEGization of m=1 5 generation lysine dendrimer-gemcitabine conjugate
Couplet VI: n=5, the PEGization of m=6 3 generation lysine dendrimer-gemcitabine conjugate
Couplet VII: a=10, the PEGization of b=1 4 generation glutamic acid dendrimer-gemcitabine conjugate
Embodiment 2
The preparation method of PEGization many peptides dendrimer drug-supplying system based on GFLG, first at gemcitabine and GFLG
Conjugate on introduce methyl ethylene, obtain conjugate MA-GFLG-GEM, standby；Again using tree-shaped greatly for the peptides as carrier
Molecule alkynyl, introduces three benzylthios on dendrimer, obtains alkynyl three benzylthio dendrimer, sloughs triphen
After methyl, there is coupling reaction with conjugate MA-GFLG-GEM, obtain the coupling of alkynyl dendrimer and gemcitabine
Thing, after through PEGization finished product.
Embodiment 3
The preparation method concrete operation step of the present invention is as follows:
The preparation of A MA-GFLG-GEM conjugate；
With Boc-GFLG-OMe as raw material, with ethylene methacrylic acyl chloride reaction prepare MA-GFLG-OH, rear MA-GFLG-OH with
DIPEA, tetrahydrothiazole-2-thion react, after reaction terminates, by product, GEMCITABINE HYDROCHLORIDE, pyrrole
Pyridine is dissolved in anhydrous dimethyl sulfoxide, and lucifuge is reacted, and reaction products therefrom is MA-GFLG-GEM conjugate；
The synthesis of B alkynyl dendrimer
With the dendrimer of Boc Yu Cbz radical protection as raw material, reacting with Pb/C under an atmosphere of hydrogen, reaction terminates
After, product is dissolved in the middle of DMF, system adds DIPEA, hexynic acid, BTA-N, N,
N', N'-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole, react under nitrogen protection, and reaction products therefrom is alkynyl
Dendrimer；
This step forms amido link by the condensation reaction of amino with carboxyl, sloughs Cbz, so that outside dendrimer
Alkynyl on shroud, provides the reaction site of click-reaction for PEGization below.
The synthesis of C alkynyl three benzylthio dendrimer
Alkynyl dendrimer is dissolved in the mixed solvent of anhydrous methylene chloride and trifluoroacetic acid, and system is protected at nitrogen
Protect lower reaction, product is dissolved in dry DMF, system adds DIPEA, 3-tri-benzylthio third
Acid, BTA-N, N, N', N'-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole, react, instead under nitrogen protection
Answering products therefrom is alkynyl three benzylthio dendrimer；
The synthesis of D alkynyl dendrimer-gemcitabine conjugate
Alkynyl three benzylthio dendrimer is dissolved in the mixed solvent of anhydrous methylene chloride and trifluoroacetic acid, system
React under nitrogen protection；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, by product
Be dissolved in the middle of dry DMF, in solution add conjugate MA-GFLG-GEM, 1,8-diazabicylo [5.4.0] 11 carbon-7-
Alkene, under nitrogen protection lucifuge reaction, reaction products therefrom is alkynyl dendrimer-gemcitabine conjugate；
E PEGization dendrimer
In a nitrogen atmosphere, by alkynyl dendrimer-gemcitabine, CuSO4·5H2O、N3-mPEG, ascorbic acid
Sodium joins DMF and H2In the middle of the mixed solvent of O, reaction system lucifuge is reacted, after reaction terminates, product is purified, dialysis,
Lyophilizing obtains PEGization dendrimer-gemcitabine conjugate.
Embodiment 4
The present embodiment is substantially the same manner as Example 3, on this basis:
Described step B, solution stirs 1h under nitrogen protection and ice bath, and is stirred at room temperature 7 days, obtains alkynyl
Dendrimer.
By controlling reaction temperature and time, can effectively reduce initial reaction rate, reduce side reaction, and can obtain
The most adorned product of the product modified completely i.e. terminal amido.
Embodiment 5
The present embodiment is substantially the same manner as Example 3, on this basis:
Described step C, reacts under nitrogen protection, and solution stirs 1h under nitrogen protection and ice bath, and in room temperature
Lower stirring 7 days, obtains alkynyl three benzylthio dendrimer.
Embodiment 6
The present embodiment is substantially the same manner as Example 3, on this basis:
Described step B, reacts under nitrogen protection, and solution stirs 60h under nitrogen protection and ice bath, and in room temperature
Lower stirring 0.5 day, obtains alkynyl dendrimer.
Described step C, reacts under nitrogen protection, and solution stirs 60h under nitrogen protection and ice bath, and in room temperature
Lower stirring 0.5 day, obtains alkynyl three benzylthio dendrimer.
Described B and step C, system reacts after terminating under nitrogen protection, and solution with ethyl acetate dilutes, and uses successively
NaHCO3、HCl、NaHCO3, NaCl washing, the anhydrous MgSO of organic facies4It is dried, and solvent, residue warp is distilled off with decompression
Recrystallization, obtains product.
Embodiment 7
The present embodiment is substantially the same manner as Example 3, on this basis:
Described D step, in solution add conjugate MA-GFLG-GEM, 1,8-diazabicylo [5.4.0] 11 carbon-
After 7-alkene, under nitrogen protection and room temperature, lucifuge is reacted 0.1 hour.
Embodiment 8
The present embodiment is substantially the same manner as Example 3, on this basis:
Described D step, in solution add conjugate MA-GFLG-GEM, 1,8-diazabicylo [5.4.0] 11 carbon-
7-alkene, under nitrogen protection and room temperature, lucifuge is reacted 90 hours, and reaction products therefrom is alkynyl dendrimer-gemcitabine
Conjugate；
Embodiment 9
The present embodiment is substantially the same manner as Example 3, on this basis:
Described D step, after reaction terminates, reactant liquor is added dropwise in the middle of ethyl acetate, and precipitate is by being centrifuged, carrying
Pure, dry, obtain alkynyl dendrimer-gemcitabine conjugate.
Embodiment 10
The preparation method of conjugate I, specifically comprises the following steps that
The preparation of A MA-GFLG-GEM conjugate
Under nitrogen protection, by Boc-GFLG-OMe(1) it is dissolved in the mixing of 10 mL anhydrous methylene chlorides and trifluoroacetic acid
In solvent (1:1, v/v) 0o24 h are stirred under C.After utilizing Rotary Evaporators to remove solvent, add appropriate absolute ether and wash
Wash three times, have white precipitate to separate out.By centrifugal collection white precipitate, and in the middle of vacuum drying oven, it is dried 0.5 h.Gained sample
Product are dissolved in the middle of the mixed solvent of 20 mL water and acetonitrile (water/acetonitrile=4/1, v/v), and by 1 M hydroxide under ice-water bath
PH value is adjusted to 9 by sodium water solution, adds 13 mL 1 M sodium hydrate aqueous solutions subsequently and continues to stir 2 h.Bathe at cryosel
Under, it is added dropwise over 0.85 mL ethylene methacrylic acyl chlorides, utilizes 1 M sodium hydrate aqueous solution to make system pH be maintained at 10 left simultaneously
Right.Add a large amount of ethyl acetate after stirring 4 h, and with 1 M aqueous hydrochloric acid solution, pH is adjusted to 2-3.Finally use saturated sodium-chloride
Solution washing three times, collects organic facies and is dried with anhydrous magnesium sulfate.Remove after solvent, mixed at ethyl acetate and normal hexane
Recrystallization in bonding solvent, obtains MA-GFLG-OH.Under nitrogen protection, by MA-GFLG-OH, DIPEA, tetrahydrochysene
Thiazole-2-thioketone is dissolved in 20 mL anhydrous methylene chlorides, and the anhydrous methylene chloride being added dropwise over EDCI under cryosel is bathed is molten
Liquid.After 24 h, in system, add 500 mL ethyl acetate, and successively with 1 M sodium bicarbonate aqueous solution, 1 M hydrochloric acid is water-soluble
Liquid, saturated sodium-chloride water solution washs.Remove solvent, be recrystallized to give bright orange in ethyl acetate with the mixed solvent of normal hexane
Color solid MA-GFLG-TT(2).By MA-GFLG-TT, GEMCITABINE HYDROCHLORIDE, pyridinium dissolution is at 30 mL anhydrous dimethyl sulfoxides
In, lucifuge is stirred at room temperature 24 h.Remove major part solvent after, frost ethyl acetate in precipitate white solid is MA-
GFLG-GEM conjugate (3) ESI-MS m/z 744 [(M+K)+, C32H41F2N7O9]
The synthesis of B alkynyl dendrimer (5)
The dendrimer (4) (3.0 g, 0.5 mmol) of Boc Yu Cbz radical protection is dissolved in 20 mL methanol,
Pb/C (2.54 g, 10%) adds in the middle of solution.Reaction system stirs 3 days under an atmosphere of hydrogen.After reaction terminates, solvent leads to
Cross decompression to be distilled off.Residue is dissolved in the middle of 50 mL DMF, DIPEA (16.8 mL, 96 mmol), hexynic acid (18
Mmol), HBTU (6.83 g, 18 mmol), HOBt (2.34 g, 18 mmol) add system, and solution is protected at nitrogen
Down and stir 1 hour under ice bath, and be stirred at room temperature 4 days.After reaction terminates, solution dilutes with 500 mL EtOAc, and
Use NaHCO successively3 aq. (satd.), 1 M HCl aq., NaHCO3Aq. (satd.), NaCl aq. (satd.) washes
Wash. the anhydrous MgSO of organic facies4It is dried, and solvent is distilled off with decompression.Residue is at the mixed solvent of ethylacetate/ether
In to be recrystallized to give white solid be alkynyl dendrimer (5), productivity: 53% (1.37g, 0.27 mmol).1H NMR
(400 MHz, DMSO): δ = 1.18-1.65 (m, CH 2-Lys and CHCCH2CH 2), 2.12-2.16 (m,
CHCCH 2 and NCH 2CH2), 2.741 (s, CHC(R)), 2.95 (s, CONHCH 2), 4.14 (s, COCH(R)NH),
6.70-7.98(m, (R)CONH); MALDI-TOF MS: m/z 5192 [(M+Na)+, C264H438N46O57Na].
The synthesis of C alkynyl three benzylthio dendrimer (6)
Alkynyl dendrimer (5) (1.0 g, 0.19 mmol) be dissolved in anhydrous methylene chloride/trifluoroacetic acid (1:
1,6 mL) in the middle of, solution is in nitrogen protection and 0oStir 24 hours under C.By decompression distillation, solvent is removed, add nothing
Water ether, separates out white precipitate.Precipitate is vacuum dried 0.5 hour and is dissolved in the middle of 30 mL dry DMF.DIPEA
(12.6 mL, 72 mmol), 3-tri-benzylthio propanoic acid (12 mmol), HBTU (4.55 g, 12 mmol), HOBt
In (1.62 g, 12 mmol) addition system, this solution stirs 1 hour under nitrogen protection and ice bath and is stirred at room temperature
48 hours.After reaction terminates, solution dilutes with 500 mL EtOAc, and uses NaHCO successively3 aq. (satd.), 1 M HCl
aq., NaHCO3Aq. (satd.), NaCl aq. (satd.) washing. the anhydrous MgSO of organic facies4It is dried, and steams with decompression
Distillate solvent.It is alkynyl triphen first sulfur that residue is recrystallized to give white solid in the mixed solvent of ethylacetate/ether
Base dendrimer (7), productivity: 75% (1.13 g, 0.14 mmol).1H NMR (400 MHz, DMSO): δ =
1.234-1.644 (m, CH 2-Lys and CHCCH2CH 2), 2.11-2.22 (m, TrtSCH 2CH 2CO, CHCCH 2 and
NCH 2CH2), 2.74 (s, CHC(R)), 2.95 (s, CONHCH 2), 4.14 (s, COCH(R)NH), 7.20-7.29
(d, ArH), 7.77-7.95 (m, (R)CONH); MALDI-TOF MS: m/z 7954 [(M+Na)+,
C468H562N46O43S12Na].
The synthesis of D alkynyl dendrimer-gemcitabine conjugate (7)
Alkynyl three benzylthio dendrimer (6) (1.19 g, 0.15 mmol) is dissolved in anhydrous methylene chloride/tri-
In the middle of Fluoroethanoic acid (1:1,8 mL), this solution stirs 0.5 hour under nitrogen protection and ice bath.When solution transfers yellow green to
After, triethyl silicane (145.6 mg, 1.25 mmol) adds system.Reaction system continues to stir under room temperature and nitrogen are protected
Mix 24 hours.By decompression distillation, solvent is removed, add absolute ether, separate out white precipitate.It is little that precipitate is vacuum dried 0.5
Time and be dissolved in the middle of 20 mL dry DMF.MA-GFLG-GEM (3,324.43 mg, 0.46 mmol), DBU (2.74
G, 18 mmol) in addition system. solution lucifuge under nitrogen protection and room temperature is reacted 72 hours.After reaction terminates, reactant liquor
It is added dropwise in the middle of 300 mL ethyl acetate, separates out red precipitate precipitate and collect by centrifugal and utilize chromatography over CC.Institute
Product is dried under vacuum, final to white solid alkynyl dendrimer-gemcitabine (7), productivity: 43%.
MALDI-TOF MS: m/z 7159 [(M+Na)+, C336H1521F6N67O72S12Na]. average with three on each target molecule
Individual GEM
E synthesis based on PEGization dendrimer-gemcitabine conjugate nano-particle
In a nitrogen atmosphere, alkynyl dendrimer-gemcitabine (7) (150 mg, 21.0 mol), CuSO4·
5H2O (90 mg, 0.36 mmol), N3-mPEG (760 mg, 0.38 mmol) and sodium ascorbate (142 mg,
0.72 mmol) add 20 mL DMF and H2In the middle of the mixed solvent of O (3:1, V/V).Reaction system lucifuge is reacted 3 days.Instead
After should terminating, solution lucifuge is dialysed 48 hours.Solvent is removed by lyophilizing, and by size exclusion chromatography post (Superose 12
HR/16/30 column on an KTA FPLC system (GE Healthcare) column) purify further.Pass through
Dialysis and lyophilizing obtain end product PEGization dendrimer-gemcitabine conjugate (8), productivity: 73 % (419.6 again
mg) MALDI-TOF MS: m/z 27372[(M+Na)+]
By in the middle of EDTA solution dialyse, after click-reaction can being removed in the middle of residued in system the copper of trace from
Son, and small molecular weight impurity, the PEG chain segment of excess and by-product simply can be removed by KTA FPLC.
Although because the molecular weight of PEG chain segment is a normal distribution, but topmost molecular weight is known, therefore may be used
The number of PEG chain segment is judged with the increase by the drawn molecular weight of mass spectrum.
3MALDI-TOF mass spectral results (27372 [(M+ according to PEGization dendrimer-gemcitabine conjugate (8)
Na)+), average each dendrimer is connected with 10 PEG chain segment.
The standard curve of the GEM that Fig. 1 is measured by utilizing HPLC, therefrom can draw PEGization dendrimer-Ji Xita
In the middle of the conjugate of shore, the drug loading of gemcitabine is 2.5%, and average each target molecule is with three GEM, and this is with above-mentioned alkynyl
The mass spectrometric data changing dendrimer-gemcitabine is identical.
Embodiment 11
The synthetic schemes of couplet II is substantially the same manner as Example 11, on this basis:
Alkynyl three benzylthio 3 generation lysine dendrimer is dissolved in anhydrous methylene chloride/trifluoroacetic acid, and system exists
The lower reaction of nitrogen protection；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, removes solvent, adds
Absolute ether, precipitate is vacuum dried, and is dissolved in the middle of dry DMF, adds the MA-GFLG-treatment of 1.05 times in solution
The factor, DBU(1,8-diazabicylo [5.4.0] 11 carbon-7-alkene), lucifuge reaction under nitrogen protection, react products therefrom
For alkynyl 3 generation lysine dendrimer-treatment factor conjugate.In a nitrogen atmosphere, by alkynyl dendrimer-control
Treat factor conjugate, CuSO4•5H2O, the N3-mPEG(nitrine methoxyl group PEG of 1.05 times of molal quantitys), sodium ascorbate joins
DMF and H2In the middle of the mixed solvent of O, reaction system lucifuge is reacted, and after reaction terminates, solution is dialysed through lucifuge, is gone by lyophilizing
Except solvent, and obtain PEGization dendrimer-gemcitabine conjugate by purification, dialysis, lyophilizing.MALDI-TOF MS:m/
z 7774 ([M+Na]+)
Embodiment 12
The synthetic schemes of couplet III is substantially the same manner as Example 11, on this basis:
Alkynyl three benzylthio 3 generation lysine dendrimer is dissolved in anhydrous methylene chloride/trifluoroacetic acid, and system exists
The lower reaction of nitrogen protection；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, removes solvent, adds
Absolute ether, precipitate is vacuum dried, and is dissolved in the middle of dry DMF, adds the MA-GFLG-treatment of 1.05 times in solution
The factor, DBU(1,8-diazabicylo [5.4.0] 11 carbon-7-alkene), lucifuge reaction under nitrogen protection, react products therefrom
For alkynyl 3 generation lysine dendrimer-treatment factor conjugate.In a nitrogen atmosphere, by alkynyl dendrimer-Ji
His shore conjugate, CuSO of west4•5H2O, the N of 10.1 times of molal quantitys3-mPEG(nitrine methoxyl group PEG), sodium ascorbate joins
DMF and H2In the middle of the mixed solvent of O, reaction system lucifuge is reacted, and after reaction terminates, solution is dialysed through lucifuge, is gone by lyophilizing
Except solvent, and obtain PEGization dendrimer-treatment factor conjugate by purification, dialysis, lyophilizing.MALDI-TOF MS:m/
z 25999 ([M+Na]+)
Embodiment 13
The synthetic schemes of couplet IV is substantially the same manner as Example 11, on this basis:
Alkynyl three benzylthio 4 generation lysine dendrimer is dissolved in anhydrous methylene chloride/trifluoroacetic acid, and system exists
The lower reaction of nitrogen protection；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, removes solvent, adds
Absolute ether, precipitate is vacuum dried, and is dissolved in the middle of dry DMF, adds the MA-GFLG-treatment of 1.05 times in solution
The factor, DBU(1,8-diazabicylo [5.4.0] 11 carbon-7-alkene), lucifuge reaction under nitrogen protection, react products therefrom
For alkynyl 4 generation lysine dendrimer-treatment factor conjugate.In a nitrogen atmosphere, by alkynyl dendrimer-control
Treat factor conjugate, CuSO4•5H2O, the N3-mPEG(nitrine methoxyl group PEG of 1.05 times of molal quantitys), sodium ascorbate joins
DMF and H2In the middle of the mixed solvent of O, reaction system lucifuge is reacted, and after reaction terminates, solution is dialysed through lucifuge, is gone by lyophilizing
Except solvent, and obtain PEGization dendrimer-treatment factor conjugate by purification, dialysis, lyophilizing.MALDI-TOF MS:m/
z 13057([M+K]+)
Embodiment 14
The synthetic schemes of couplet V is substantially the same manner as Example 11, on this basis:
Alkynyl three benzylthio 5 generation lysine dendrimer is dissolved in anhydrous methylene chloride/trifluoroacetic acid, and system exists
The lower reaction of nitrogen protection；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, removes solvent, adds
Absolute ether, precipitate is vacuum dried, and is dissolved in the middle of dry DMF, adds the MA-GFLG-treatment of 1.05 times in solution
The factor, DBU(1,8-diazabicylo [5.4.0] 11 carbon-7-alkene), lucifuge reaction under nitrogen protection, react products therefrom
For alkynyl 5 generation lysine dendrimer-treatment factor conjugate.In a nitrogen atmosphere, by alkynyl dendrimer-control
Treat factor conjugate, CuSO4•5H2O, the N of 2.1 times of molal quantitys3-mPEG(nitrine methoxyl group PEG), sodium ascorbate joins
DMF and H2In the middle of the mixed solvent of O, reaction system lucifuge is reacted, and after reaction terminates, solution is dialysed through lucifuge, is gone by lyophilizing
Except solvent, and obtain PEGization dendrimer-treatment factor conjugate by purification, dialysis, lyophilizing.MALDI-TOF MS:m/
z 25571([M+H]+)
Embodiment 15
The synthetic schemes of couplet VI is substantially the same manner as Example 11, on this basis:
Alkynyl three benzylthio 3 generation lysine dendrimer is dissolved in anhydrous methylene chloride/trifluoroacetic acid, and system exists
The lower reaction of nitrogen protection；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, removes solvent, adds
Absolute ether, precipitate is vacuum dried, and is dissolved in the middle of dry DMF, in solution add 6.1 times MA-GFLG-treatment because of
Son, DBU(1,8-diazabicylo [5.4.0] 11 carbon-7-alkene), lucifuge reaction under nitrogen protection, reaction products therefrom is
Alkynyl 3 generation lysine dendrimer-treatment factor conjugate.In a nitrogen atmosphere, by alkynyl dendrimer-treatment
Factor conjugate, CuSO4•5H2O, the N3-mPEG(nitrine methoxyl group PEG of 5.03 times of molal quantitys), sodium ascorbate join DMF
And H2In the middle of the mixed solvent of O, reaction system lucifuge is reacted, and after reaction terminates, solution is dialysed through lucifuge, is removed molten by lyophilizing
Agent, and obtain PEGization dendrimer-treatment factor conjugate by purification, dialysis, lyophilizing.MALDI-TOF MS:m/z
19416([M+K]+)
Embodiment 16
The synthetic schemes of glutamic acid dendrimer-gemcitabine coupling is even with lysine dendrimer-gemcitabine
The synthetic schemes (embodiment 10 15) of connection is essentially identical.
Embodiment 17
The synthetic method of Boc-GFLG-OMe
The most under nitrogen protection, (11,10.5 g), and (12,8.6 g), HOBT for H-Phe-OMe hydrochlorate for Boc-Gly-OH
(10.808 g), HBTU (30 g), DIPEA (48 mL) be dissolved in the middle of 50 mL dry DMF, and solution is 0o2 are stirred under C
H, is then stirred at room temperature 24 h.Reaction system is added in the middle of 500 mL ethyl acetate after terminating by reaction, and uses successively
NaHCO3Aq. (satd.), 1 M HCl aq., NaHCO3Aq. (satd.), NaCl aq. (satd.) washing. organic facies
Use anhydrous MgSO4It is dried, and solvent is distilled off with decompression.Residue is recrystallization in the mixed solvent of ethylacetate/ether
Obtain white solid Boc-GF-OMe.ESI-MS m/z 359[(M+Na)+] productivity: 85% yield.
The most under nitrogen protection, Boc-Leu-OH (14.0 g), H-Gly-OMe hydrochlorate (5.0 g), HOBT (10.8
G), EDC (16.0 g), DIPEA (48.8 mL) are dissolved in 50 mL dry DMF.Solution is 0oStir 2 h under C, then exist
24 h are stirred under room temperature.Reaction system is added in the middle of 500 mL ethyl acetate after terminating by reaction, and uses NaHCO successively3 aq.
(satd.), 1 M HCl aq., NaHCO3Aq. (satd.), NaCl aq. (satd.) washing.The anhydrous MgSO of organic facies4
It is dried, and solvent is distilled off with decompression.Residue is recrystallized to give white solid in the mixed solvent of ethylacetate/ether
Boc-LG-OMe。ESI-MS m/z 341[(M+K)+] productivity: 87% yield.
3.Boc-GF-OMe (9.0 g, 27.0 mmol) is initially dissolved in proper amount of methanol, then adds at 0 DEG C
Enter 1 M NaOH aq. (80.0 mL) solution and be stirred at room temperature 3 h, then with 1 M HCl aq. by pH regulator to 2-3.
Reaction system is added in the middle of 500 mL ethyl acetate after terminating by reaction, and washs three times with NaCl aq. (satd.).Decompression
Solvent is distilled off.Gained white solid is Boc-GF-OH, standby.
The most under nitrogen protection, Boc-LG-OMe (8.607 g, 28.5 mmol) is initially dissolved in 20 mL anhydrous two
Chloromethanes is central (1:1, V/V) with the mixed solvent of trifluoroacetic acid, and this mixed system is 0o24 h are stirred under C.Steamed by decompression
Evaporate and solvent is removed, add absolute ether, separate out white precipitate.Precipitate is vacuum dried 0.5 hour and to be dissolved in 50 mL anhydrous
In the middle of DMF.DIPEA (19.5 mL), Boc-GF-OH (6.0 g), HBTU (10.6 g), HOBT (3.8 g) are sequentially added into
System under nitrogen protection and 0oStir 1 h under C, be then stirred at room temperature 24 h.React reaction system after terminating
Add in the middle of 500 mL ethyl acetate, and successively with NaHCO3 aq. (satd.), 1 M HCl aq., NaHCO3 aq.
(satd.), NaCl aq. (satd.) washing. organic facies is dried with anhydrous MgSO4, and solvent is distilled off with decompression.Residual
Thing is recrystallized to give white solid Boc-GFLG-OMe in the mixed solvent of ethylacetate/ether.ESI-MS m/z 529
[(M+Na)+] productivity: 70.3% yield.
Embodiment 18
In the embodiment of the present invention 10, the synthetic method of Boc (12)-G3-Cbz (12)
The most under nitrogen protection, Boc-Lys (Boc)-OH (3.523 g, 10.17 mmol), HOBT (3.865 g,
10.17 mmol), HBTU (3.856 g, 10.17 mmol), DIPEA (7.1 mL, 40.68 mmol) are dissolved in 50 mL
In the middle of dry DMF, and 0oC stirs 30 min.2.42 g tri-(2-amine ethyl) after amine is added dropwise over this system at room temperature stir
Mix 24 h.Reaction system is added in the middle of 500 mL ethyl acetate after terminating by reaction, and uses NaHCO successively3Aq. (satd.),
1 M HCl aq., NaHCO3Aq. (satd.), NaCl aq. (satd.) washing. the anhydrous MgSO of organic facies4It is dried, and uses
Decompression is distilled off solvent.It is 1 generation to rely ammonia that residue is recrystallized to give white solid in the mixed solvent of ethylacetate/ether
Acid dendrimer (G1).1H NMR (400 MHz, DMSO):δ=1.15–1.75 (m, CH 2-Lys and CH 3-Boc),
.40 (m, NCH 2CH2NHCO), 2.80 (m, CH2NH-Lys), 3.08 (m, NCH2CH 2NHCO),177 3.78–4.0
(m, COCH(R)NH), 6.70 (s, NHCO), 7.61 (s, NHCO); MALDI-TOF MS:178 m/z 1153.75
[(M+Na)+, C54H102N10O15Na] productivity: 87.5%
The most under nitrogen protection, 1 generation lysine dendrimer (2.26 g, 2mmol) be initially dissolved in 20 mL without
Water dichloromethane is central (1:1, V/V) with the mixed solvent of trifluoroacetic acid, and this mixed system is 0o24 h are stirred under C.By subtracting
Solvent is removed by pressure distillation, adds absolute ether, separates out white precipitate.Precipitate is vacuum dried 0.5 hour and is dissolved in 50 mL
In the middle of dry DMF.DIPEA (4.89 mL, 28 mmol), Boc-Lys (Boc)-OH (2.42,7 mmol), HBTU
(2.65 g, 7 mmol), HOBT (0.945 g, 7 mmol) is sequentially added into system under nitrogen protection and 0oStir under C
Mix 1 h, be then stirred at room temperature 24 h.Reaction system is added in the middle of 500 mL ethyl acetate after terminating by reaction, and successively
With NaHCO3 aq. (satd.), 1 M HCl aq., NaHCO3 aq. (satd.), NaCl aq. (satd.) washs.Organic
It is dried with anhydrous MgSO4, and solvent is distilled off with decompression.Residue is heavily tied in the mixed solvent of ethylacetate/ether
It was 2 generations lysine dendrimer (G2) that crystalline substance obtains white solid.1H NMR (400 MHz, DMSO):δ=1.15–1.80
(m, CH 2-Lys and CH 3-Boc), 2.42 (m, NCH 2CH2NHCO), 2.80–3.15 (m, CH 2NH-Lys and
NCH2CH2NHCO), 3.80–4.22 (m, COCH(R)NH), 6.40 (s, NHCO), 6.78 (s, NHCO), 6.90
(s, NHCO), 7.78 (s, NHCO); MALDI-TOF MS: 2522.64 [(M+Na)+,C120H222N22O33Na]. productivity:
73%
The most under nitrogen protection, 2 generation lysine dendrimers (2.0 g, 0.8 mmol) are initially dissolved in 20 mL
Anhydrous methylene chloride is central (1:1, V/V) with the mixed solvent of trifluoroacetic acid, and this mixed system is 0o24 h are stirred under C.Pass through
Solvent is removed by decompression distillation, adds absolute ether, separates out white precipitate.Precipitate is vacuum dried 0.5 hour and is dissolved in 50
In the middle of mL dry DMF.DIPEA (7.9 mL, 60 mmol), Cbz-Lys (Boc)-OH (4.256,11.2 mmol),
HBTU (4.244 g, 11.2 mmol), HOBT (1.512 g, 11.2 mmol) are sequentially added into system and protect at nitrogen
Lower and 0oStir 1 h under C, be then stirred at room temperature 24 h.React and after terminating, reaction system is added 500 mL acetic acid second
In the middle of ester, and successively with NaHCO3 aq. (satd.), 1 M HCl aq., NaHCO3 aq. (satd.), NaCl aq.
(satd.) washing.Organic facies is dried with anhydrous MgSO4, and solvent is distilled off with decompression.Residue is in ethylacetate/ether
Mixed solvent in be recrystallized to give white solid be 3 generations lysine dendrimer (G3).MALDI-TOF MS: m/z
5668[(M+Na)+, C288H438N46O69] productivity: 68%
The raw material sources of synthetic method of the present invention: Boc-Gly-OH, H-Phe-OMe hydrochlorate, Boc-Leu-OH, H-Gly-
OMe hydrochlorate, Boc-Lys (Boc)-OH, Cbz-Lys (Boc)-OH all buys from Shanghai gill biochemistry company limited；Three (2-
Amine ethyl) buy from TCI chemical company.
Embodiment 19
The present invention has carried out following experiment and has detected dendrimer-gemcitabine couplet I:
One, size, pattern and Zeta potential
The present invention utilizes Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK)
Hydrodynamic size and Zeta potential to nano-particle are characterized.PEGization dendrimer-gemcitabine conjugate
It is dissolved in 10 mL redistilled waters to obtain the aqueous solution of 100 μ g/mL.This aqueous solution is dropped on copper mesh, disperses a few minutes
After, unnecessary solvent filter paper is wiped away and the most naturally dries with prepared TEM sample.Will based on PEGization dendrimer-
The nano-particle aqueous solution (100 μ g/mL) of gemcitabine couplet directly drops on silicon chip and the most naturally dries, and
Further its size is characterized by FE-SEM.
Dynamic light scattering (DLS) result (Fig. 2) shows that PEGization dendrimer-gemcitabine conjugate can be in water
It is gathered into the nano-particle of 109.2 nm.The driving force of this self assembly behavior come from the hydrophilic of PEG chain segment with tree-shaped greatly
Balance between molecular hydrophobicity.It addition, the self assembly behavior that gemcitabine contained in the middle of system is also whole system carries
Supplyπ-πStacking, the negative charge of the driving force nano grain surface such as dipole effect and hydrogen bond action, can reduce nano-particle with
Strong effect between serum albumin, thus reduce macrophage and nano-particle is swallowed, extend its circulation time in vivo.
Therefore, this system is obtained in that in the middle of tumor tissues preferably assembles, thus demonstrates preferable antitumous effect.
Utilizing TEM that the pattern of nano-particle has been carried out further sign, result shows that the size of nano-particle is 100
About nm (Fig. 3).The result of comprehensive DLS, nano-particle based on PEGization dendrimer-gemcitabine couplet has 90 ~
The size of 110 nm, this feature can make full use of EPR effect and assemble at tumor tissues, can effectively prolong again simultaneously
Its circulation time in vivo long, thus be expected to show good antitumous effect.
Utilizing DLS and TEM to characterize the pattern of the nano-particle of couplet V, DLS result shows nano-particle
A size of 129.5nm, TEM result shows that the size of nano-particle is at about 110 nm.The result of comprehensive DLS, sets based on PEGization
The nano-particle of shape macromole-gemcitabine couplet V has the size of 100 ~ 130 nm.
Utilizing DLS and TEM to characterize the pattern of the nano-particle of couplet VI, DLS result shows nano-particle
A size of 82.5nm, TEM result shows that the size of nano-particle is at about 90 nm.The result of comprehensive DLS, tree-shaped based on PEGization
The nano-particle of macromole-gemcitabine couplet VI has the size of 80 ~ 100 nm.
Two, tumor cell line kind, cell is cultivated and zoopery
1, TC-1 cell line is bought from Chinese Academy of Sciences's Shanghai cell bank, and with RPMI 1640 as culture medium, with 10%(v/
V) heat inactivated foetal calf serum and 1% penicillin and streptomycin mixture are supplement, at the gas of 5% carbon dioxide/95% air
Atmosphere and 37oCultivate under conditions of C.
Take the logarithm TC-1 cell more than trophophase, with cell count after 0.05% trypsinization, use fresh cultured
Liquid is configured to 1 × 105/ml, and 96, each hole of orifice plate adds 100uL, is placed in 37 DEG C, cultivates in 5 % CO2 incubators.Purification
PEGization 3 generation dendrimer-gemcitabine conjugate and gemcitabine sample PBS dissolve, filtration sterilization.24 h
After add the PEGization 3 generation dendrimer-gemcitabine conjugate of variable concentrations of equimultiple dilution or gemcitabine sample (
20uL/ hole), same concentration sets 5 multiple holes, and negative control hole adds 20uL culture medium.After continuing to cultivate 48 h, every hole
Add 100uL CCK-8 solution, after putting room temperature 2 h, survey absorbance at 450 nm by the Well full-automatic microplate reader of scan MK,
Calculating killing rate as follows: killing rate (%)=[control wells D (450)-experimental port D (570)]/control wells D
(570) × 100 %, by calculating the inhibition percentage of cell growth, go out PEGization 3 generation by Orig n 6.0 computed in software
The IC50 (half-inhibition concentration) of dendrimer-gemcitabine conjugate and gemcitabine respectively 1.52 ug/mL, 0.04
Ug/mL, result is shown in Fig. 4.
2, BALB/c mouse breast cancer 4T1 cell strain and African green monkey kidney cell strain COS-7 are from Chinese Academy of Sciences's Shanghai cell bank
Buy.37 DEG C, 5% CO2Atmosphere under, cell is RPMI 1640 culture medium (10%(V/V) heat-inactivated fetal bovine serum, 1% dish Buddhist nun
XiLin/streptomycin) in monolayer culture.Female BAl BIc/c mice (20 ± 2 g, 6-8 week old) is purchased from Chengdu Da Shuo biotech firm
, and raise in constant temperature, day alternates with night environment.
Vitro cytotoxicity is tested
Non-medicine carrying carrier, PEG-G3 (Lys)-GFLG-gemization 3 generation dendrimer-gemcitabine conjugate) with
And the cytotoxicity of GEMCITABINE HYDROCHLORIDE free drug (GEM HCl) utilizes CCK-8 method to be measured.In 96 orifice plates, often
Hole adds 5.0 × 103Individual 4T1 or COS-7 cell.After 24 hours, by non-medicine carrying carrier, PEG-G3 (Lys)-GFLG-gem with
And the sterile water solution of GEM HCl is separately added in reserved aperture.Continue to cultivate 48 hours, remove culture medium and every hole adds
100 L 10% CCK-8/ RPMI 1640(V/V) mixed solution.Orifice plate continues to cultivate 2 hours at 37 DEG C, utilizes microplate reader
The cell survival rate of each sample is measured by (ThermoFisher SCIENTIFIC).
Result is as it is shown in figure 5, the IC of PEG-G3 (Lys)-GFLG-gem50(cell half lethal concentration) is 31.2 ng/mL,
Slightly above 11.3 ng/mL of GEMCITABINE HYDROCHLORIDE free drug.As having the drug delivery vehicle of nano-scale, PEG-
G3 (Lys)-GFLG-gem needs to be entered in the middle of lysosome by endocytosis, then discharges medicine, and this allows for it and acts as
The free drug being entered cell by free diffusing it is slower than by speed.This is probably its cytotoxicity less than free drug.Meanwhile,
Non-medicine carrying carrier in the case of high concentration (4.0-107.8 μ g/mL, Fig. 6) does not demonstrate the obvious poison for 4T1 cell
Property；It addition, under 323.2 μ g/mL concentration conditions, non-medicine carrying carrier is still the thinnest for COS-7 normal cell
Cellular toxicity.Result above shows that non-medicine carrying carrier has preferable biocompatibility at cellular layer mask, and medicine-carried system is thin simultaneously
Cellular toxicity is provided by the medicine discharged.
Internal animal experiment
At every BALB/c mouse subcutaneous injection 50 μ L 4T1 cell PBS suspension (5 × 105) with swollen in setting up 4T1 body
Tumor model.After about two weeks, when gross tumor volume rises to 100-150 mm3Time, by tumor-bearing mice according to injection drug species not
With being randomly divided into three groups: normal saline group (Saline, matched group), GEM HCl (1 mg GEM/kg) and PEG-G3
(Lys)-GFLG-gem(1 mg GEM/kg).Within every four days, pass through tail vein injection 200 μ L test solution, and every two days measure
Gross tumor volume and Mouse Weight.Gross tumor volume calculates according to below equation:V (mm3) = 1/2 × length (mm)
× width (mm2). after 21 days, disconnected neck puts to death all mices, peels off tumor and weighs, calculates tumour inhibiting rate (TGI).TGI=
(1-(treatment group average tumor weight)/(matched group average tumor weight)) × 100%
Result as it is shown in fig. 7, the therapeutic effect of GEMCITABINE HYDROCHLORIDE free drug is not satisfactory, its 21 days Relative tumor
Rate of growth is 483.21 ± 104.64 %, and PEG-G3 (Lys)-GFLG-GEM treatment group obtained phase at 21 days in contrast
It is 86.17 ± 38.27 % to tumor growth rate.After 21 days, disconnected neck is put to death all tumor-bearing mices and peels off tumor, enters tumor
Row is weighed.As shown in Figure 8,9, the tumor of GEMCITABINE HYDROCHLORIDE free drug group is heavily 1924 ± 504 mg (TGI=44.59
%), and the tumor of PEG-G3 (Lys)-GFLG-GEM treatment group is heavily 350 ± 139 mg, and its tumour inhibiting rate TGI is 89.92%.This
Show the anti-tumor in vivo effect free drug to be far superior to of medicine carrying delivery system.Superior internal of nano drug-carrying delivery system
It is poly-that antitumous effect and biocompatibility are probably its surface institute's long circulating time electronegative, internal, higher tumor locus
Collection degree and its enzyme sensitive medicaments release behavior cause jointly.
Claims (8)
1. the preparation method of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine, it is characterised in that: it is administered
System is antitumor drug gemcitabine, target function sex factor GFLG and the conjugate of PEGization peptides dendrimer, its knot
Structure is as follows:
Wherein, the structure of A is:
B is 3 ~ 5 generation peptides dendrimers of PEGization, and the terminal amino group of dendrimer is connected with group A；
On the gemcitabine conjugate with GFLG, first introduce methyl ethylene, obtain conjugate MA-GFLG-GEM, standby；Again
Using the peptides dendrimer alkynyl as carrier, dendrimer introduces three benzylthios, obtain alkynyl triphen first
, after sloughing trityl, there is coupling reaction with conjugate MA-GFLG-GEM, obtain alkynyl tree-shaped in sulfenyl dendrimer
The conjugate of macromole and gemcitabine, after through PEGization finished product.
The preparation of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine the most according to claim 1
Method, it is characterised in that: the end of described peptides dendrimer connects altogether 1 or multiple described A group.
The preparation of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine the most according to claim 1
Method, it is characterised in that: the described peptides dendrimer that peptides dendrimer is lysine or glutamic acid.
The preparation of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine the most according to claim 1
Method, it is characterised in that: the grain diameter of described PEGization peptides dendrimer targeting drug delivery system is 80 ~ 130 nm.
The preparation of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine the most according to claim 1
Method, it is characterised in that: concrete operation step is as follows:
The preparation of A MA-GFLG-GEM conjugate
With Boc-GFLG-OMe as raw material, prepare MA-GFLG-OH, rear MA-GFLG-OH and N, N-with ethylene methacrylic acyl chloride reaction
Diisopropylethylamine, tetrahydrothiazole-2-thion react, after reaction terminates, by molten to product, GEMCITABINE HYDROCHLORIDE, pyridine
Solution is in anhydrous dimethyl sulfoxide, and lucifuge is reacted, and reaction products therefrom is MA-GFLG-GEM conjugate；
The synthesis of B alkynyl dendrimer
With the dendrimer of Boc Yu Cbz radical protection as raw material, react with Pb/C under an atmosphere of hydrogen, after reaction terminates, will
Product is dissolved in the middle of DMF, adds DIPEA, hexynic acid, BTA-N, N, N', N'-in system
Tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole, react under nitrogen protection, and reaction products therefrom is that alkynyl is tree-shaped greatly
Molecule；
The synthesis of C alkynyl three benzylthio dendrimer
Alkynyl dendrimer is dissolved in the mixed solvent of anhydrous methylene chloride and trifluoroacetic acid, and system is under nitrogen protection
Reaction, is dissolved in product in dry DMF, adds DIPEA, 3-tri-benzylthio propanoic acid, benzene in system
And triazole-N, N, N', N'-tetramethylurea hexafluorophosphate, I-hydroxybenzotriazole, react under nitrogen protection, react institute
Obtaining product is alkynyl three benzylthio dendrimer；
The synthesis of D alkynyl dendrimer-gemcitabine conjugate
Alkynyl three benzylthio dendrimer is dissolved in the mixed solvent of anhydrous methylene chloride and trifluoroacetic acid, and system is at nitrogen
React under gas shielded；After solution transfers yellow green to, system adds triethyl silicane, after reaction terminates, product is dissolved
In the middle of dry DMF, in solution add conjugate MA-GFLG-GEM, 1,8-diazabicylo [5.4.0] 11 carbon-7-alkene,
The lower lucifuge reaction of nitrogen protection, reaction products therefrom is alkynyl dendrimer-gemcitabine conjugate；
E PEGization dendrimer
In a nitrogen atmosphere, by alkynyl dendrimer-gemcitabine conjugate, CuSO4 5H2O, N3-mPEG, Vitamin C
Acid sodium joins in the middle of the mixed solvent of DMF and H2O, and reaction system lucifuge is reacted, and after reaction terminates, product is purified, saturating
Analysis, lyophilizing obtain PEGization dendrimer-gemcitabine conjugate.
The preparation of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine the most according to claim 5
Method, it is characterised in that: described B and step C, system reacts after terminating under nitrogen protection, and solution with ethyl acetate dilutes,
And wash with NaHCO3, HCl, NaHCO3, NaCl successively, organic facies is dried with anhydrous MgSO4, and is distilled off molten with decompression
Agent, residue, through recrystallization, obtains product.
The preparation of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine the most according to claim 5
Method, it is characterised in that: described D step, in solution add conjugate MA-GFLG-GEM, 1,8-diazabicylo [5.4.0]
After 11 carbon-7-alkene, under nitrogen protection and room temperature, lucifuge is reacted 0.1 ~ 90 hour.
The preparation of the PEGization peptides dendrimer targeting drug delivery system of load gemcitabine the most according to claim 5
Method, it is characterised in that: described D step, after reaction terminates, reactant liquor is added dropwise in the middle of ethyl acetate, and precipitate leads to
Cross and be centrifuged, purify, be dried, obtain alkynyl dendrimer-gemcitabine conjugate.
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| CN106729749B (en) * | 2017-01-24 | 2019-11-12 | 四川大学 | It is a kind of to can be realized the transmission system and the preparation method and application thereof that gene and drug carry altogether |
| CN106880848B (en) * | 2017-02-14 | 2019-09-06 | 四川大学 | Biodegradable poly HPMA-Gd magnetic resonance imaging probe and preparation method thereof |
| CN107625968B (en) * | 2017-09-20 | 2020-09-29 | 四川大学 | Tumor specific tissue-cell double-permeation nanoparticle, preparation method and application thereof |
| CN111514309B (en) * | 2019-02-03 | 2021-09-17 | 重庆阿普格雷生物科技有限公司 | Polyethylene glycol coupled drug, preparation method and application thereof |
| CN110183504B (en) * | 2019-06-13 | 2023-07-07 | 湖南大学 | Gemcitabine prodrug with tumor targeting function and preparation method and application thereof |
| CN110270372A (en) * | 2019-06-21 | 2019-09-24 | 三峡大学 | The preparation method and application of gold nano grain |
| CN110591075B (en) * | 2019-06-28 | 2022-03-04 | 四川大学华西医院 | PEG-Peptide linear-tree-shaped drug delivery system and preparation method and application thereof |
| CN111298132B (en) * | 2020-02-22 | 2022-06-24 | 新乡医学院 | Tree-shaped molecule gemcitabine self-assembled nano prodrug and preparation method and application thereof |
| CN115068624B (en) * | 2022-06-27 | 2024-02-13 | 重庆理工大学 | Peptide dendrimer drug-carrying system and preparation method and application thereof |
| CN118459755B (en) * | 2024-04-29 | 2024-10-11 | 复旦大学 | Gemcitabine nano prodrug with stable circulation and preparation method and application thereof |
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| 《The potential of self-assembled, pH-responsive nanoparticles of mPEGylated peptide dendronedoxorubicin conjugates for cancer therapy》;Wenchuan She et al.;《Biomaterials》;20121126;第34卷;1-9 * |
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