CN104650194B - A kind of peptide dendrimer drug and its preparation method and application - Google Patents
A kind of peptide dendrimer drug and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of peptide dendrimer drugs and its preparation method and application, belong to field of biomedicine, and the drug includes peptide dendrimer skeleton and end group functional group.Guarantee the biological activity of drug by the end group functional group, the peptide dendrimer periphery can be grafted a large amount of end group functional group, to effectively amplify the biological effect of end group functional group by the enlarge-effect of dendrimer.The drug has accurate molecular structure, and surface functional group abundant, good water solubility, also has class globulin and easily by Biological characteristics such as cellular uptakes, the biology for enriching peptide dendrimer should have and promote the development of peptide medicament.The preparation method, simple process, and the structure of peptide dendrimer drug can be accurately controlled by controlling the outfit of each raw material.
Description
Technical field
The invention belongs to fields of biomedicine, and in particular to a kind of therapeutic type peptide dendrimer anticancer drug.
Technical background
As the problems such as environmental pollution, food safety is on the rise, the disease incidence of the major diseases such as malignant tumour significantly increases
Height, disease treatment cause the extensive concern of people.Currently, the chemotherapy based on chemicals is current most effective disease
One for the treatment of method；Tumour cell is acted on by drug molecule to realize the purpose of disease treatment.However, the exploitation of new drug and
Using the great difficult problem for being all the time treatment of cancer research, but the exploitation of new drug is to improve medication effect, reduce poison pair
Effect, the great strategy for realizing multi-medicine tolerant reversal.
China is the production of maximum drug and big export country in the world, but wherein imitation medicine production accounts for 97% or more.For
Realize that, from medical big country to the developing goal of medicine power, the research and development of reinforcing original new drug is imperative.Country is logical
Cross the development that various policies, science and technology item etc. push " great new drug initiative ", medicine of encouraging innovations research and development, guidance new drug industrialization.
Up to the present, invention possesses the significant challenge that the new drug of independent intellectual property right is still China's medicament research and development.New target is visited
The MOLECULE DESIGN of rope and new drug is the important breakthrough mouth of new drug development.
In recent years, the research and development of natural product extraction, prodrug and metal class drug is fruitful in cancer treatment.
Strikingly, the advantage based on the peptide medicament of natural amino acid in antineoplaston constantly highlights, such as its specificity
Action target spot, efficient biological activity and low toxic side effect.Researchers are just attempting to have not by designing and constructing
With the peptide medicament of amino acid sequence and molecular structure to obtain efficient anti-tumor drug.Currently, the weight that peptide medicament faces
Big challenge is high production cost, synthesis and purifying difficulty is big, poorly water-soluble, the low main problem of cellular uptake amount；Therefore it opens
Hair is a kind of have be easily-synthesized, efficient peptide medicament is of great significance to new drug development.
Summary of the invention
Structure based on peptide dendrimer is accurate, the spy of polyfunctionality, biological activity and bionic function etc.
Point, peptide dendrimer have the potential quality as a kind of novel peptide type drug.It is anti-swollen to develop a kind of therapeutic type dendrimer
Tumor medicine is not only to have expanded the exploitation of peptide kind new medicine from the angle of molecular structure, while also perfect dendrimer is in biology
The research of medical domain.The content of present invention had both been different from functional development of the previous carrier model dendrimer to drug loading,
It is different from the development of previous line style peptide medicament again, is a kind of peptides new drug development innovated in structure and composition.In this base
On plinth, the present invention provides a kind of therapeutic type peptide dendrimer anticancer drugs, are imitated using the amplification of peptide dendrimer
The biological function of the bio-active group in amino acid should effectively be amplified, and then realize the purpose of disease treatment.
The present invention is achieved through the following technical solutions:
A kind of therapeutic type peptide dendrimer drug, including peptide dendrimer skeleton and end group functional group.
Guarantee the biological activity of drug by the end group functional group, the peptide dendrimer periphery can be grafted largely
End group functional group, to effectively amplify the biology of end group functional group by the enlarge-effect of dendrimer
Effect.
Alternately, in above-mentioned peptide dendrimer drug, the end group functional group is with biology
Learn active amino acid.The biological function and interaction of biomacromolecules characteristic of amino acid are efficiently used, and amino acid is residual
Base can improve physical and chemical properties of drugs (such as dissolubility), convenient for playing therapeutic effect in vivo.Amino acid had both been used as end group function
Change the branching unit that group is used as peptide dendrimer skeleton again, keeps peptide dendrimer medicines structure more compact and smart
Really, have be easily-synthesized, the characteristics such as easy purification, good water solubility.
Alternately, in above-mentioned peptide dendrimer drug, the end group functional group contains indole ring.
Indole ring can interfere cell week using the enlarge-effect and nucleic acid supermolecular mechanism of dendrimer with nucleic acid interaction
Its anti-tumor activity may be implemented in phase.
Alternately, in above-mentioned peptide dendrimer drug, the end group functional group is tryptophan.Benefit
The indole ring in tryptophan is used to cause cell to wither using the indole ring and nucleic acid supermolecular mechanism of tryptophan as active group
It dies, to realize the treatment of tumour；Drug water solubility can be enhanced in amino acid residue；Tryptophan is also used as peptides tree-shaped big simultaneously
A part of branching unit of molecular skeleton.
Alternately, in above-mentioned peptide dendrimer drug, the peptide dendrimer is with amino acid
For a generation for branching unit or two generations or three generations or four generation peptide dendrimers.
Alternately, the branch in above-mentioned peptide dendrimer drug, in the peptide dendrimer skeleton
Change unit is lysine, in serine, glutamic acid, arginine, histidine, threonine, tyrosine, aspartic acid, tryptophan
It is at least one.
Alternately, in above-mentioned peptide dendrimer drug, the peptide dendrimer is fan-shaped or ball
Shape.Make the structure of the drug concrete kind globulin using peptide dendrimer.
Alternately, in above-mentioned peptide dendrimer drug, the structural formula of the peptide dendrimer
It is as follows:
Wherein Rm represents the core element of global molecular, and m represents the degree of functionality of core element, and the degree of functionality of core element can
Think 3,4,6 or 8；K is amino acid branching unit, and G 1.O and G 2.0 respectively represents a generation and two generation peptide dendrimers；n
Represent end group functional group.
Alternately, in above-mentioned peptide dendrimer drug, the core element is One of.
The present invention also provides a kind of preparation methods for preparing the peptide dendrimer drug, which is characterized in that
The following steps are included:
(1) peptide dendrimer is prepared；
(2) end group functional group is grafted in peptide dendrimer periphery.
Alternately, the preparation of peptide dendrimer described in step (1) can use divergent method or convergence method
Or the method that divergent-convergent combines.
Alternately, the preparation method of peptide dendrimer described in step (1) specifically:
A) protective group is carried out to amino acid: according to the core element surface official of peptide dendrimer to be prepared
The difference that can be rolled into a ball protects amino acid, protects if core element surface functional group is amino to the amino of amino acid
Shield protects the carboxyl of amino acid if core element surface functional group is hydroxy or carboxy；
B) it prepares generation dendrimer: weighing branching core (degree of functionality n, n > 1), the amino containing blocking group in proportion
Sour (1.5n equivalent), condensing agent (1.5n equivalent), catalyst (1.5n equivalent) and organic base (4n equivalent), in 0 DEG C of nitrogen protection
Under the conditions of, solvent is added and carries out dehydration condensation；Then it reacts at room temperature, after reaction, acquired solution is washed, does
It is dry, it is concentrated under reduced pressure, precipitation and separation is obtained with blocking group first generation peptide dendrimer in organic solvent；
C) it is deprotected: accurately weighing first generation peptide dendrimer, it is molten that solvent is added in deprotecting regent (20n equivalent)
Solution reacts at room temperature 4 hours, deprotection under nitrogen protection, is concentrated under reduced pressure, and by extracting or precipitating, obtains first generation peptides
Dendrimer；
D) two generation dendrimers are prepared: weighing first generation peptide dendrimer (degree of functionality 2n) in proportion, containing guarantor
Amino acid (3n equivalent), condensing agent (3n equivalent), catalyst (3n equivalent) and the organic base (8n equivalent) for protecting group, in 0 DEG C of nitrogen
Under the conditions of gas shielded, solvent is added and carries out dehydration condensation；Then it reacts at room temperature, after reaction, acquired solution warp
Washing, it is dry, it is concentrated under reduced pressure, precipitating is obtained with blocking group second generation peptide dendrimer in organic solvent；It repeats
The above deprotection and step of condensation, can be obtained third, forth generation dendrimer.
Alternately, end group functional group is connect by the condensation reaction of amino and carboxyl in the step (2)
Branch is peripheral to peptide dendrimer.
Alternately, when the end group functional group of the peptide dendrimer drug is with biological activity
Amino acid (such as tryptophan) when, it is directly that the amino acid with biological activity is outermost as peptide dendrimer skeleton
The branching unit of layer pair prepares the dendrimer in higher generation, can be realized in the step (2) outside peptide dendrimer
Enclose the purpose of grafting end group functional group.
The present invention also provides a kind of above-mentioned peptide dendrimer drug application in preparations of anti-tumor drugs.It will
Peptide dendrimer drug of the present invention has good inhibiting effect to tumour as a kind of anticancer drug.
All features disclosed in this specification or disclosed all methods or in the process the step of, in addition to mutually exclusive
Feature and/or step other than, can combine in any way.
Beneficial effects of the present invention:
The invention proposes a kind of new therapeutic type peptide dendrimer anticancer drugs, successfully solve peptide medicament
Poorly water-soluble, wear membrane permeability difference and the problems such as high production cost, be easily-synthesized, good water solubility and anti-tumor activity it is high
Peptide dendrimer anticancer drug.
Peptide dendrimer anticancer drug of the present invention has accurate molecular structure, surface function abundant
Group also has class globulin and easily by Biological characteristics such as tumour cell intakes, enriches the biology of peptide dendrimer
There should be and promote the development of peptide medicament.
In optional way of the invention, uses tryptophan for end group functional amino acid, utilize putting for dendrimer
Big effect can make the indole ring of tryptophan and the biology function of nucleic acid interaction and then performance peptide dendrimer drug
Energy.
Preparation method of the present invention, simple process, and peptide can be accurately controlled by controlling the outfit of each raw material
The structure and function of class drug.
Detailed description of the invention
Fig. 1 is the schematic diagram of the working principle of peptide dendrimer drug of the present invention.
Fig. 2 is the synthetic route chart of peptide dendrimer drug described in the embodiment of the present invention 2.
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of peptide dendrimer drug described in the embodiment of the present invention 2.
Fig. 4 is the mass spectrum of peptide dendrimer drug described in the embodiment of the present invention 2.
Fig. 5 is the reversed-phase high performance liquid chromatography figure of peptide dendrimer drug described in the embodiment of the present invention 2.
Fig. 6 is the agarose gel electrophoresis of peptide dendrimer drug described in the embodiment of the present invention 3 and DNA effect.
Fig. 7 is the fluorescence emission spectrum of peptide dendrimer drug described in the embodiment of the present invention 3 and DNA effect.
Fig. 8 is the transmission electron microscope photo of peptide dendrimer drug described in the embodiment of the present invention 3 and DNA effect.
Fig. 9 is the uv-vis spectra of peptide dendrimer drug described in the embodiment of the present invention 3 and DNA effect.
Figure 10 is the hydrogen spectrum of peptide dendrimer drug described in the embodiment of the present invention 3 and DNA effect.
Figure 11 is the infared spectrum of peptide dendrimer drug described in the embodiment of the present invention 3 and DNA effect.
Figure 12 is peptide dendrimer drug described in the embodiment of the present invention 4 to the cell survival of different tumour cells
Rate.
Figure 13 is the laser co-focusing picture of peptide dendrimer drug described in the embodiment of the present invention 4 and DNA effect.
Figure 14 is the laser co-focusing picture of the intake of peptide dendrimer drug cell described in the embodiment of the present invention 4.
Figure 15 is the cell cycle distribution of peptide dendrimer drug described in the embodiment of the present invention 4 and DNA effect.
Figure 16 is gross tumor volume and the changes of weight that BABL/c mouse treats 21 days in the embodiment of the present invention 5.
Figure 17 is that tumor tissues immunohistochemistry is sliced photo and half after the treatment of BABL/c mouse 21 days in the embodiment of the present invention 5
Quantitative data analysis.
Specific embodiment:
Above content of the invention is described in further detail again by the following examples.But this should not be understood
Range for the above-mentioned theme of the present invention is only limitted to example below.Do not depart from done within the spirit and principles in the present invention it is any
Modification, and the equivalent replacement or improvement made according to ordinary skill knowledge and customary means should all be included in this
In the protection scope of invention.
Embodiment 1: the preparation of peptide dendrimer
A) protective group is carried out to amino acid: according to the core element surface official of peptide dendrimer to be prepared
Can the difference of group amino acid is protected, if core element surface functional group is amino or hydroxyl to the amino of amino acid into
Row protection protects the carboxyl of amino acid if core element surface functional group is carboxyl；
B) it prepares generation dendrimer: weighing branching core (degree of functionality n, n > 1), the amino containing blocking group in proportion
Sour (1.5n equivalent), condensing agent (1.5n equivalent), catalyst (1.5n equivalent) and organic base (4n equivalent), at 0 DEG C, nitrogen protection
Under the conditions of, solvent is added and carries out dehydration condensation；Then it reacts at room temperature, after reaction, acquired solution is washed dry
It is dry, it is concentrated under reduced pressure, is obtained with organic solvent deposit with blocking group first generation peptide dendrimer；
C) it is deprotected: accurately weighing first generation peptide dendrimer, it is molten that solvent is added in deprotecting regent (20n equivalent)
Solution reacts at room temperature 4 hours, deprotection under nitrogen protection, is concentrated under reduced pressure, and by extracting or precipitating, obtains first generation peptides
Dendrimer；
D) two generation dendrimers are prepared: weighing first generation peptide dendrimer (degree of functionality 2n) in proportion, containing guarantor
Amino acid (3n equivalent), condensing agent (3n equivalent), catalyst (3n equivalent) and the organic base (8n equivalent) for protecting group, at 0 DEG C, nitrogen
Under the conditions of gas shielded, solvent is added and carries out dehydration condensation；Then it reacts at room temperature, after reaction, acquired solution warp
Washing, it is dry, it is concentrated under reduced pressure, organic solvent deposit is obtained with blocking group second generation peptide dendrimer；
The above deprotection and step of condensation are repeated, third, forth generation dendrimer can be obtained.
It chooses respectively One of be used as branching core,
Using one or more of lysine, arginine, histidine, glutamic acid or aspartic acid, tryptophan as branching unit, it is made
A series of peptide dendrimer in one to four generations.Its structural formula is as shown in Equation 1.
Condensing agent described in the present embodiment, catalyst, organic base, solvent may be selected well known in the prior art for carboxylic
Various condensing agents, catalyst, the organic base, solvent of the dehydrating condensation of base and amino.
A kind of embodiment 2: specific synthesis example of peptide dendrimer drug (synthetic route such as Fig. 2)
The synthesis of generation peptide dendrimer (G1-Poss-Lys)
30mL concentrated hydrochloric acid is added drop-wise in 350mL methanol at 50 DEG C, continues to increase the temperature to 90 DEG C, three second of 3- aminopropyl
15 mL of oxysilane is slowly added dropwise, and for 24 hours, tetrahydrofuran precipitating obtains eight poly- (3- aminopropyl) silsesquioxane salt to confined reaction
Sour (PossHCl).1H NMR(600 MHz,DMSO-d6)δ8.14(s,2H),3.61-3.59(t,2H),1.69(m,2H),
0.75-0.72(t,2H)。
Weigh poly- (3- aminopropyl) the silsesquioxane heptane hydrochloride (PossHCl) of 2.0g cage modle eight, three nitrogen of 8.0g O- benzo
Azoles-tetramethylurea hexafluorophosphoric acid ester (HBTU) and 2.5g I-hydroxybenzotriazole (HOBT) are added in the mono- neck bottle of 100mL, will
5.0g Boc-lys (Boc)-OH is added in 50mL constant pressure funnel, vacuumizes, inflated with nitrogen.About 40mL weight is added with syringe
The n,N-diisopropylethylamine (DIPEA) of 3mL is added under ice bath stirring for steamed DMF solvent, continues to remove after stirring half an hour
Ice bath is changed to react 48h at room temperature.After solvent is removed under reduced pressure, chloroform dissolution is added, successively with saturation NaHCO3、NaHSO4、
NaCl washing.With anhydrous MgSO4After drying, rotation removes solvent, recrystallizes in acetonitrile, obtains white powdery solid G1-Poss-
Lys-Boc。1H NMR(600 MHz,DMSO-d6)δ7.76(s,1H),6.73(s,2H),3.83(d,1H),3.02-2.86(d,
4H), 1.46-1.19 (m, J=7.0 Hz, 26H), 1.37 (s, 6H), 0.55 (s, 2H).
Deprotection
By the G1-Poss-Lys-Boc white powder of 1.0g, the trifluoroacetic acid (TFA) of 6mL is added, reacts at room temperature 6h.Subtract
TFA is removed in pressure rotation, is drained with oil pump, and anhydrous ether stirring, which is added, white precipitate generation, obtains compound G1-Poss-Lys.It produces
Object is directly used in the next step without purification.
The synthesis of peptide dendrimer drug (G2-Poss-Trp)
The synthesis step for repeating G1-Poss-Lys-Boc, by poly- (3- aminopropyl) the silsesquioxane salt of cage modle eight therein
Acid changes generation peptide dendrimer (G1-POSS-Lys) into, changes Boc-lys (Boc)-OH into Boc-Trp-OH, synthesizes G2-
Poss-Trp-Boc。1H NMR(600MHz,DMSO-d6)δ10.76(s,2H),8.32(s,3H),7.84-6.66(m,10H),
4.24-4.16(t,3H),3.02(m,8H),1.27-1.11(s,26H),0.55(s,2H)。
Deprotection and purifying
By the white powder of the G2-Poss-Trp-Boc of 1.0g, the trifluoroacetic acid of 2mL is added, reacts at room temperature 6h.Decompression rotation
TFA is removed, is drained with oil pump, anhydrous ether stirring, which is added, white precipitate generation, obtains compound G2-Poss-Trp.It will obtain
G2 peptide dendrimer be dissolved in deionized water, dialyse 3 days, be freeze-dried it is spare to get to periphery be tryptophan peptide
Class dendrimer drug (G2-Poss-Trp).
It is shown through magnetic resonance detection (see Fig. 3) and a generation and two generation peptide dendrimers is successfully prepared by this method
(nuclear magnetic spectrum that Fig. 3 is G2-Poss-Trp-Boc).To be further determined to synthesized compound, mass spectrum is also used
Instrument (U.S. Waters public affairs Q-TOF premier) is determined the molecular weight of gained compound.As a result also confirm that gained produces
Object is target compound (mass spectrogram that Fig. 4 is G2-Poss-Trp).It is tree-shaped that the peptides further are had rated with high performance liquid chromatography
The purity of macromolecular drug is up to 99.9% (see Fig. 5).
Embodiment 3: to the peptide dendrimer drug prepared in embodiment 2, the detection of following several respects is carried out to it:
(1) ability that different amounts of peptide dendrimer drug and DNA effect are observed with gel electrophoresis retardation experiment, will
The peptide dendrimer drug and p-EGFPC1 Plasmid DNA (including 200ng DNA) of different W/P ratios (ratio is 0.1~40)
Room temperature compound 30min, W/P ratio refers to phosphate group (P) molar ratio in tryptophan (W) and DNA in peptide medicament.Compound warp
1% agarose gel electrophoresis 100mV 45min is crossed, ethidium bromide staining is observed under the ultraviolet lamp of 254nm, and Fig. 6 result is aobvious
Show, when the molar ratio of peptide dendrimer drug and p-EGFPC1 plasmid is greater than 10:1, can effectively with p-EGFPC1 matter
Grain interaction, blocks its movement.
(2) simultaneously, fluorescence spectrum further illustrates between peptide dendrimer drug (TRPDs) and p-EGFPC1 plasmid
There are supermolecular mechanisms.Firstly, by peptide dendrimer drug, (concentration is 5 μ g/ according to different W/P ratios (from 20:1 to 1:1)
ML it) with the compound 30min of DNA room temperature, is sent out by the fluorescence that F-7000 sepectrophotofluorometer detects peptide dendrimer drug
Penetrate spectrum change.As shown in fig. 7, in the case where peptide dendrimer drug concentration is certain (5 μ g/mL), with amount of DNA
It is continuously increased, peptide dendrimer drug and DNA interact, and tryptophan modules is caused to be quenched in the characteristic absorption peak of 365nm
It goes out, peptide dendrimer and DNA form aggregation and the absorption peak for scattering light 578nm caused to enhance.
(3) for the interaction further probed between the peptide dendrimer drug and nucleic acid, firstly, by saturating
Radio mirror (TEM) characterizes the nanoscale of peptide dendrimer medicament nucleic acids aggregation.By peptide dendrimer drug
(concentration is 100 μ g/mL) and DNA according to W/P be the compound 30min of 10 room temperatures after be lyophilized, pass through tem observation peptide dendrimer
The aggregation that drug and DNA are formed.As shown in figure 8, be 100 μ g/mL, W/P molar ratios in peptide dendrimer drug being 10
When, the aggregation of peptide dendrimer drug and DNA formation about 60nm.
(4) secondly, ultraviolet-visible absorption spectroscopy shows that there are π-phase interactions between peptide dendrimer drug and DNA
With.Firstly, by peptide dendrimer drug, (concentration is 10 μ g/mL) and the room DNA according to different W/P ratios (from 5:1 to 30:1)
The peptide dendrimer drug (TRPDs) of temperature compound 30min, not compound DNA pass through UV, visible light spectrophotometric as control
The uv-vis spectra of meter detection peptide dendrimer drug changes and then studies the π-of peptide dendrimer drug and DNA
Interaction.As shown in figure 9, in the case where peptide dendrimer drug concentration is certain (10 μ g/mL), with amount of DNA
Be continuously increased, since there are pi-interactings with DNA for the tryptophan indole ring of peptide dendrimer, make the ultraviolet of tryptophan
The absorption peak for absorbing 250-300nm broadens, and peak value increases.
(5) in addition, by peptide dendrimer drug and DNA difference W/P (such as 100:1 and 25:1) compound 30min of room temperature,
The pi-interacting of peptide dendrimer drug and DNA is studied in freeze-drying by nuclear-magnetism.As shown in Figure 10, nuclear-magnetism result
There are pi-interactings with DNA for the tryptophan indole ring for showing in the peptide dendrimer drug, with peptide dendrimer
The nuclear magnetic spectrogram of drug is compared, and with the increase of DNA, the chemical shift of proton of indole ring is obviously mobile to High-Field, while peak shape
It broadens.
(6) cold finally, by peptide dendrimer drug and the compound 30min of DNA difference W/P (such as 10:1 and 1:1) room temperature
Be lyophilized it is dry, we by infrared spectroscopy (Figure 11) as can be seen that peptide dendrimer drug can with DNA interact cause
Guanine stretching vibration is from 1698cm-1It is displaced to 1675cm-1,PO2Symmetrical stretching vibration is from 1088cm-1It is displaced to 1016cm-1.
Embodiment 4: biological assessment
CCK-8 method measures peptide dendrimer vitro Drug toxicity test.
Selection in the active growth phase kinds of tumor cells (selection 4T1, HepG2, HeLa, MCF-7, MCF-7/ADR,
7 kinds of tumor cell lines such as SKOV3 and SKOV3/ADR) it is inoculated in 96 orifice plates, after culture 24 hours, it is added in embodiment 2 and is made
Peptide dendrimer drug, be divided into 4 groups, administration concentration is respectively 10ug/mL, 50ug/mL, 70ug/mL, 100ug/mL,
6 Duplicate Samples are arranged in each experimental group, while following control group is arranged: changing the peptide dendrimer drug into color respectively
Propylhomoserin, generation lysine, tryptophan/generation lysine mixture and two generation lysines, remaining operation are identical.Continue culture one
After fixing time, after being rinsed with PBS buffer solution (pH 7.4) and the more culture medium that renews, CCK-8 is added, is protected from light and continues culture 2h,
With the light absorption value at microplate reader measurement 490nm, cell survival rate is calculated.Experimental result is as shown in figure 12, and the peptides are tree-shaped big
Molecular drug all has efficient anti-tumor activity to various tumour cells, and anti-tumor activity increases with concentration and enhanced.It is right
Show that tryptophan, generation lysine, tryptophan/generation lysine mixture and two generation lysines have good according to group experimental result
Good biocompatibility.
Change in fluorescence in cell in vitro.
Tumour cell is taken, culture medium dilution is added, is inoculated in the ware of glass bottom by the density of every hole fixed number, is pasted to cell
After wall growth, the peptide dendrimer drug (blue-fluorescence) that concentration is 10 μ g/mL is added.After cell culture 2 hours, use
After PBS (pH 7.4) is rinsed 2 times, 33342 marker DNA of Hoechst (blue-fluorescence), after being rinsed 2 times with PBS (pH 7.4), then
PBS is added, with the change in fluorescence situation of peptide dendrimer drug in laser co-focusing observation cell in vitro.Experimental result is such as
Shown in Figure 13, in the case where 405nm excitation, it can not only emit at low band (400nm to 500nm) (a and b in Figure 13) glimmering
Light, equally higher band (550nm to 800nm) (c and d in Figure 13) emit fluorescence, show low band emit fluorescence by
Peptide dendrimer generates, and the fluorescence of high band transmitting has peptide dendrimer drug to lead with intracellular DNA interaction
Scattered light intensity is caused to increase, the phenomenon and external peptide dendrimer drug are consistent with DNA complex fluorescence spectrum change, table
The bright peptide medicament can interact really with intracellular DNA.
Cell in vitro intake experiment.
Tumour cell is taken, culture medium dilution is added, is inoculated in the ware of glass bottom by the density of every hole fixed number, is pasted to cell
After wall growth, it is the peptide dendrimer drug (green fluorescence) that 10 μ g/mL FITC is marked that concentration, which is added,.Cell culture is several
After a time point, after being rinsed 2 times with PBS (pH 7.4), 33342 marker DNA of Hoechst (blue-fluorescence), with PBS (pH
7.4) after rinsing 2 times, PBS is added, with the cell of laser co-focusing observation cell in vitro intake peptide dendrimer drug
Interior distribution and situation of change.Laser co-focusing experimental result (Figure 14) shows that peptide dendrimer drug can effectively enter carefully
Born of the same parents, and interact with nucleic acid substances intracellular, to realize its antitumous effect.
Apoptosis detection.
Tumour cell is taken, culture medium dilution is added, is inoculated in six orifice plates by the density of every hole fixed number, is pasted to cell
After wall growth, the peptide dendrimer drug that concentration is 10 μ g/mL is added and collects cell, and with 70% after culture 24 hours
Ethyl alcohol is fixed for 24 hours, centrifugation, and after PBS is rinsed one time, cell carries out PI and dyes 30min, centrifugation, after PBS is rinsed one time, with PBS weight
Outstanding cell, the influence of peptide medicament cell cycle is observed by flow cytometer.Experimental result is as shown in figure 15, the peptides tree
Shape macromolecular drug can make cell cycle arrest in the sub-G1 phase, and tryptophan, generation lysine, tryptophan/generation rely ammonia
Acid blend and two generation lysines all do not cause the change of cell cycle, peptide dendrimer drug can by with core
Sour supermolecular mechanism, and then the cell cycle is influenced, eventually lead to the apoptosis of cell.
Embodiment 5: anti-tumor experiment in animal body.
4T1 tumor model is established in 4 week old BABL/c mouse (about 20-25g), it is long to 100cm to tumour3When, by mouse
Peptide dendrimer drug and control group G2-Lys are configured to a certain concentration by random grouping, and every mouse carries out muscle note
It penetrates, every 100 μ L, carries out the administration of tail vein every three days, be administered four times altogether, while every 2 days to tumor size and small
Mouse weight record, observation period are 21 days.Mouse is dissected after experiment, prepares vital tissue and slices of organs.Experimental result
As shown in Figure 16,17 and 18, Figure 16 A is internal anti-tumor experiment arrangement, and Figure 16 B is the tumour relative volume within 21 days observation periods
The variation of size, Figure 16 C are the variation of the tumour relative body weight within 21 days observation periods, and Figure 17 A is tumor biopsy immunohistochemistry (H&
E), CD-31, Ki-67 and TUNEL dyeing, Figure 17 B are tumor tissue section CD-31 staining positive cells semi-quantitative analysis, figure
17C is tumor tissue section Ki-67 staining positive cells semi-quantitative analysis, and Figure 17 D is tumor tissue section TUNEL stained positive
Cell semi-quantitative analysis.The peptide dendrimer drug can effectively inhibit tumour growth, new vessels, tumour increment, and
Cause the apoptosis of tumour cell, meanwhile, mouse body in order, does not cause the lesion of other organs.
Embodiment 6
WithAs branching core, using tryptophan as branching unit, the peptide in one to four generation is made
Class dendrimer.It is tree-shaped to the peptides in resulting one to four generation big according to biotinylated biomolecule evaluation method as described in example 4
Molecular drug carries out antitumous effect detection.As the result is shown: the peptide dendrimer drug in one to four generation all has good
Water solubility wears membrane permeability and antitumous effect, and with the increase of algebra, antitumous effect enhancing.Due to the therapeutic type peptide
The inner cavity of class dendrimer and end group have both indole ring structures, can further enhance the supermolecular mechanism of itself and nucleic acid molecules
(conjugation, intercalation and electrostatic interaction etc.), therefore its antitumous effect can be enhanced.
Embodiment 7
It choosesIt is condensed, is obtained with indoles with carboxylic indole ring (such as 3- indolepopionic acid)
Ring is 0 generation dendritic macromole drug of end group functional group.It choosesAs branching core, with
Amino acid is branching unit, 1,2,3 generation dendrimer skeletons is made respectively, respectively by the amino of each generation macromolecular skeleton periphery
With carboxylic indoles cyclic condensation, 1,2,3 generation dendrimer drugs are obtained.
Embodiment 8
It choosesIt is condensed, is obtained using indole ring as end group with carboxylic indole ring (such as 3- indolepopionic acid)
0 generation dendrimer drug of functionalization group.It choosesAs branching core, using amino acid as branching unit,
1,2,3 generation dendrimer skeletons are made respectively, respectively by the amino of each generation macromolecular skeleton periphery and carboxylic indole ring
Condensation, obtains 1,2,3 generation dendrimer drugs.
According to biotinylated biomolecule evaluation method as described in example 4, the peptide in 0 to 3 generation resulting to embodiment 7 and 8 respectively
Class dendrimer drug carries out antitumous effect detection.The peptide dendrimer drug in resulting 0 to 3 generation of embodiment 7 and 8
Result it is essentially identical: with the raising of algebra, peripheral trp residue number is stepped up；Therefore, tree-shaped big point of peptides
The binding ability of son and nucleic acid enhances, and anti-tumor capacity steps up；As algebra increase causes indoles residue to increase, enhance
A possibility that dendrimer and amino acid act on, but reduces the water solubility of dendrimer, antitumous effect, which is weaker than, to be contained
There is the peptide dendrimer of trp residue.
The above description is only a preferred embodiment of the present invention, is merely illustrative for the purpose of the present invention, and not restrictive；
Those of ordinary skill in the art understand, carry out many changes to it in the spirit and scope defined by the claims in the present invention,
Modification or even equivalent change, but fall within protection scope of the present invention.
Claims (9)
1. a kind of therapeutic type peptide dendrimer drug, it is characterised in that: including peptide dendrimer skeleton and end group function
Group can be changed, the end group functional group is the amino acid with biological activity, it can guarantee the biological activity of drug,
The end group functional group contains indole ring, and the peptide dendrimer periphery is grafted a large amount of end group functional group,
To which the enlarge-effect by dendrimer effectively amplifies the biological effect of end group functional group, the end group function
Change group is tryptophan.
2. therapeutic type peptide dendrimer drug according to claim 1, which is characterized in that tree-shaped big point of the peptides
Son is using amino acid as a generation for branching unit or two generations or three generations or four generation peptide dendrimers.
3. therapeutic type peptide dendrimer drug according to claim 1, which is characterized in that tree-shaped big point of the peptides
Branching unit in son is lysine, serine, glutamic acid, arginine, histidine, threonine, tyrosine, aspartic acid, color
At least one of propylhomoserin.
4. therapeutic type peptide dendrimer drug according to claim 1, which is characterized in that tree-shaped big point of the peptides
Son is spherical shape.
5. therapeutic type peptide dendrimer drug according to claim 4, which is characterized in that the peptides are tree-shaped big
The structural formula of molecule is as follows:
(formula 1)
Wherein Rm represents the core element of global molecular, and m represents the degree of functionality of core element, and the degree of functionality of core element can be
3,4,6 or 8；K is amino acid branching unit, and G 1.0 and G 2.0 respectively represent a generation and two generation peptide dendrimers；N generation
Table end group functional group.
6. therapeutic type peptide dendrimer drug according to claim 5, which is characterized in that the core element is(n=3),(n=4),(n=4), (n=6)、One of (n=8).
7. the preparation method of any one of therapeutic type peptide dendrimer drug in a kind of such as claim 1-6,
It is characterized in that, comprising the following steps:
(1) peptide dendrimer is prepared；
(2) end group functional group is grafted in peptide dendrimer periphery.
8. preparation method according to claim 7, which is characterized in that pass through amino and carboxyl in the step (2)
End group functional group is grafted to peptide dendrimer periphery by condensation reaction.
9. a kind of therapeutic type peptide dendrimer drug as any one of in claim 1-6 is preparing antineoplastic
Application in object.
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