CN105853355B - A kind of bookbinding peptide polymer micellar preparation for anticancer - Google Patents
A kind of bookbinding peptide polymer micellar preparation for anticancer Download PDFInfo
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Abstract
The invention belongs to field of pharmaceutical preparations, are related to polypeptide delivery system, and in particular to one kind is contained with the active bookbinding peptide polymer micellar preparation of specific anti-cancer.This delivery system is made of target function material, amphipathic nature block polymer and bookbinding peptide, it can restore the active bookbinding peptide of cancer suppressor protein p53 by containing, it is delivered in cancer cell and plays its activity for inhibiting growth of cancer cells, target anticancer effect is improved, and more preferable with anticancer effect after Temozolomide drug combination.The present invention passes through In vitro and in vivo activity and evaluates, it was demonstrated that the delivery system can successfully will be in bookbinding delivery of peptides to target area and entrance target cell, with specific therapeutic effect.
Description
Technical field
The invention belongs to technical field of medicine, are related to a kind of polypeptide formulations, and in particular to one kind, which contains, has specificity
The polymer micelle preparation of the bookbinding peptide of anticancer activity.
Background technique
Cancer remains the number one killer for threatening human health, although based on the conventional therapy means based on radiation and chemotherapy
It is achieved periodic result between the past few decades, but its limited curative effect and serious toxic side effect to explore cancer hair
Anttdisease Mechanism, related target and effort a moment for specific target spot and Mechanism Design specific drug did not also stop.Study people
Member with accumulation, gradually specifies that multiple and cancer occurs, shifts closely related access by long-term research in recent years, adjoint
The discovery of the related important target spot of a variety of cancers, determined basis for the research and development pad of specific anti-cancer drugs.
Cancer suppressor protein p53 and its negativity regulatory protein MDM2 and MDMX are that the carcinogenesis gradually illustrated in recent years is led to
Road, and a brand-new target spot is provided for anticancer research.Studies have shown that p53 gene is the most important tumor suppressor gene of the mankind, by
It expresses the specific actor that the p53 albumen generated is its cancer suppressing function.The generation of cancer and p53 gene and albumen have very
Important connection.Research is it is also shown that there are about the expression of the intracellular p53 protein normal of 50% cancerous tissue, but are expressed due to high
The negativity regulatory protein such as MDM2, so that p53 albumen is combined and loses tumor-suppression activity.If can be for p53 and MDM2 albumen
Combination design competition inhibitor, then the p53 being combined can be released again and play its effect, to reach anticancer
Purpose, this has treatment of for example some soft tissue cancers of malignant tumour of high expression negativity regulatory protein, glioma etc.
Highly important meaning.
In recent years, in the research in relation to cancer related target, gradually by mainly studying all kinds of factors and cell outside cancer cell
Associated receptor is changed into potential target molecule in cancer cell on film, and the formation of this research variation tendency, mainly has benefited from point
The rise and development of sub- pharmacy.It is thin only to enter cancer for any specific drug or molecular entity for target spot in cancer cell
It is intracellular really to play a role, and to reach this target, it is necessary first to drug or molecular entity are effectively delivered to cancer group
Knit position.
There is research to screen to obtain a kind of polypeptide (PMI) for being directed to MDM2 and MDMX, warp by mirror image display technique of bacteriophage
Preliminary identification can combine negativity regulatory protein MDM2 and MDMX, thus it is speculated that its with potential inhibition MDM2 and MDMX combination p53 simultaneously
Restore the anticancer activity of p53 function；But described PMI polypeptide itself does not have the function into cell, needs suitable delivering system
System, which carries it into, plays its anticancer activity in cancer cell.Cell membrane is worn by imparting it after bookbinding peptide technology modification PMI polypeptide
Ability, but its poorly water-soluble, activity is low in environment containing serum, and in-vivo tumour targeting is poor.Polymer micelle has been at present
It is widely used in the formulation development of chemotherapeutics, genomic medicine and diagnostic medicine (substances such as radioactivity, paramagnetic) etc., certain grain
There is diameter polymer micelle good passive targeting can assign its long circulating and active targeting etc. by the modification to material
Function.
The active bookbinding peptide of p53 can be restored by being contained using polymer micelle, and play grinding for its specific anti-cancer effect
Study carefully and has no similar report both at home and abroad.
Summary of the invention
Present invention aims at the defects for making up the prior art, provide a kind of polypeptide polymer micella system for anticancer
A kind of agent, and in particular to polymer micelle preparation contained with the active bookbinding peptide of specific anti-cancer.
Specifically, the bookbinding peptide polymer micellar preparation for anticancer of the invention, which is characterized in that by target function
Material, amphipathy macromolecule block copolymer and bookbinding peptide composition, in which: target function material is as one group of polymer micelle
At partially target cell, amphipathy macromolecule block copolymer are used to contain and pass as polymer micelle skeleton part for identification
Bookbinding peptide is sent, bookbinding peptide, which has, restores cancer suppressor protein p53 active function, is delivered and can inhibit cancer cell life in cancer cell
It is long, realize the targeted therapy of cancer.
In the present invention, target function material is the targeted molecular and amphiphilic block that can specifically bind cancer cell
The active targeting compound of object material coupling, amphipathy macromolecule block copolymer material therein are selected from: polyethylene glycol
Lactic acid (PEG-PLA), polyethylene glycol-lactic-co-glycolic acid (PEG-PLGA), polyethylene glycol-distearoylphosphatidyl second
Hydramine (PEG-DSPE), polyethylene glycol-dipalmitoylphosphatidylcholine (PEG-DPPC) etc..Polyethylene glycol end group is active group
Group can be coupled with the targeted molecular of specific binding target cell.Active group on polyethylene glycol is mainly selected from Malaysia acyl
Imido grpup, sulfydryl, amide groups, amino, carboxyl, biotin or Avidin.
In the present invention, the targeted molecular that can specifically bind cancer cell or target cell includes: specific binding cancer cell
Or target cell smaller ligand such as folic acid (having tumour cell targeting affinity characteristic), oxidation ascorbic acid (have brain blood capillary
Endothelial cell, tumour cell target affinity characteristic) etc.；Polypeptide ligand such as RGD peptide (is arginine, glycine and aspartic acid
The peptide molecule of conservative sequence has tumour cell, endothelial cells in tumor neogenetic blood vessels targeting affinity characteristic comprising linear,
The polypeptide of annular and backward D configuration), LyP-1 peptide (is the peptide molecule with 9 amino acid residue CGNKRTRGC, has swollen
Oncocyte, tumor-associated macrophage, tumor lympha endothelial cell target affinity characteristic comprising linear, annular and backward D
The polypeptide of configuration), CDX peptide (is the peptide molecule with 16 amino acid residue FKESWREARG, has brain capillary endothelium
Cell-targeting affinity characteristic comprising linear polypeptide and backward D configuration polypeptide) etc., and it is directed to cancer cell or target cell surface
Special target carries out the polypeptide aglucon that phage display screens, and is such as the sequence that human glioma U87 is screened
The aptamer that the polypeptide aglucon or SELEX technology screening of VTWTPQAWFQWV obtains is such as thin for human prostata cancer LNCaP
The aptamer A10PSMA Apt etc. that cellular surface antigen PSMA is screened；Antibody such as anti-HER2 etc..
In the present invention, the bookbinding peptide of delivering is screened to obtain to carry out mirror image display technique of bacteriophage for cancer protein MDM2
Polypeptide or derivatives thereof, be capable of the combination of competitive antagonism MDM2/MDMX and cancer suppressor protein p53, restore p53 protein active.
Binding peptide or derivatives thereof includes the peptides such as PMI polypeptide, PMI-N8A, sequence TSFAEYWN-LLSP, TSFAEYWALLSP or
TSFAEYWALLS, setting site is 4,11.
The present invention is the carrier for binding peptide with polymer micelle, obtains carrying peptide polymer micella using film forming aquation method, and
Through gel column separating purification, bookbinding peptide polymer micellar preparation is obtained；Its partial size is characterized by particle size determination instrument, and is counted
Calculate the encapsulation rate of polypeptide in said preparation；The bookbinding peptide polymer micellar preparation that the present invention is used for anticancer is made, constructs a kind of energy
Enough by the bookbinding delivery of peptides of anticancer to the polymer micelle delivery system in target area and its target cell.
In the present invention, the polymer micelle partial size for containing bookbinding peptide is 30-200nm.It can be sent out by intravenously administrable approach
Wave the effect for the treatment of cancer.
The present invention is by following step, using human glioma cell U87 as model, has carried out external (cell) to its activity
With (subcutaneous tumors and brain primary tumor) pharmacodynamic evaluation in vivo.
1) target function material is prepared
It is reacted by the free sulfhydryl groups of polypeptide with the amphipathy macromolecule block copolymer material of maleimide activation, such as
Maleimide-polyethylene glycol-polylactic acid, maleimide-polyethylene glycol-distearoyl phosphatidyl ethanolamine realize membrane material
Synthesis；
2) polymer micelle preparation of preparation bookbinding peptide
The polymer micelle for ordering peptide is carried using film forming aquation method preparation, and through gel column separating purification, obtains bookbinding peptide
Polymer micelle preparation.Its partial size is characterized by particle size determination instrument, and calculates the encapsulation rate of polypeptide in said preparation；
3) the Anticancer Activity in vitro evaluation of peptide polymer micella is bound
It is carried out by anticancer activity of the external model to bookbinding peptide stapled PMI polymer micelle of human glioma U87
Evaluation, evaluation means are cancer cell survival rate after mtt assay measurement administration, and the cancer cell survival rate drawn under different dosing concentration becomes
Change curve；
4) the anticancer Evaluation on specificity (mechanism of action) of peptide polymer micella is bound
Bookbinding peptide stapled PMI polymer micelle is carried out by model of the human glioma U87 cell of p53 wild type
Anticancer mechanism research, using the relevant three kinds of important fingers of p53 access intracellular after the detection administration of Western Blotting technology
Mark the changes of contents situation of (p53, MDM2 and p21)；
5) anti-cancer activity in vivo for binding peptide polymer micella evaluates (subcutaneous tumors)
Using U87 subcutaneous transplantation tumor nude mouse as animal pattern, to the internal anti-of bookbinding peptide stapled PMI polymer micelle
Cancer activity is evaluated, and the curve evaluation therapeutic effect that knurl product relative value changes over time is drawn；
6) Anticancer Activity in vitro for binding peptide polymer micella evaluates (brain primary tumor)
Using U87 brain primary tumor nude mouse as animal pattern, to the internal anticancer of bookbinding peptide stapled PMI polymer micelle
Activity is evaluated, and evaluation index is the life span of lotus knurl nude mouse.
The results show that preparation of the present invention can specifically kill the human glioma U87 cell of p53 wild type,
The growth of U87 subcutaneous tumors can be significantly inhibited and extend the life span of U87 brain primary tumor animal pattern, prompt the present invention to have good
Good potentiality use for cancer treatment.
The present invention has the advantages that will have specific anti-cancer using amphipathic nature block polymer as main carriers material
The novel bookbinding peptide of mechanism contains in polymer micelle, the potentiality with good treating cancer.The present invention utilizes bookbinding peptide
Strategy and polymer micelle cancer target strategy, can effectively overcome the serum stability under physiological environment, wear cell membrane ability
With tumor tissues selectivity, after can ensureing that polypeptide is delivered to tumor locus, reach stable anticancer effect into tumour cell,
It has broad application prospects.
Detailed description of the invention
The nuclear-magnetism H stave of Fig. 1: RGD- polyethylene glycol-polylactic acid is levied
Wherein, nuclear-magnetism H spectrum display, compared with maleimide-polyethylene glycol-polylactic acid, has been free of Malaysia in product
Acid imide characteristic peak δ amine ppm) 6.7, illustrate that reaction carries out completely, efficient liquid phase is not detected in product containing free polypeptide, says
Bright dialysis is more thorough.
Fig. 2: bookbinding peptide polymer micellar preparation inhibits cancer cell effect picture in vitro
Wherein, bookbinding peptide polymer micellar preparation is the RGD-PEG-PLA polymer micelle (RGD for containing bookbinding peptide sPMI
The active target preparation of cyclic peptide modification), respectively with empty vectors RGD-PEG-PLA micella, free bookbinding peptide sPMI and small molecule
P53-MDM2 inhibitor Nutlin-3 is external living with the MTT cytoactive detection method evaluation of human glioma U87 as control
Property.
Fig. 3: bookbinding peptide polymer micellar preparation specific in vitro inhibits cancer cell mechanism choice
Which show the U87 cells for selecting p53 wild type protein, are separately added into the RGD cyclic peptide modification of various dose
After binding peptide polymer micellar preparation, using free bookbinding peptide sPMI and small molecule p53-MDM2 inhibitor Nutlin-3 as pair
According to measuring the feelings of tri- kinds of albumen changes of contents of p53, MDM2 and p21 in two kinds of cells by Western Blotting technology
Condition.
Fig. 4: anticancer effect figure (subcutaneous tumors) in bookbinding peptide polymer micellar preparation body
Which show using subcutaneous lotus knurl (human glioma U87) nude mice as activity in vivo evaluation model animal, 48 lotuses
Tumor nude mice is randomly divided into 6 groups (n=8), respectively physiological saline group, and polypeptide sPMI group of dissociating contains the RGD- of bookbinding peptide sPMI
PEG-PLA polymer micelle low dosage (4mg/kg) and high dose group (10mg/kg), Temozolomide group (50mg/kg, take orally) and
Administering drug combinations group (50mg/kg Temozolomide adds 10mg/kg to bind peptide polymer micella) is changed over time with knurl product relative value
Curve evaluation therapeutic effect, and compare the difference of each group and negative control group (*: p < 0.05 have significant difference；*: p
< 0.01 has very significant difference；*: p < 0.001 of * has especially significant difference).
Fig. 5 shows anticancer effect figure (brain primary tumor) in bookbinding peptide polymer micellar preparation body,
Wherein, using the tumor bearing nude mice in situ of people's glioma U87 as activity in vivo evaluation model animal, 50 tumor bearing nude mices with
Machine is divided into 5 groups (n=10), respectively physiological saline group, contains the RGD-PEG-PLA polymer micelle (10mg/ of bookbinding peptide sPMI
Kg), Temozolomide group (50mg/kg takes orally), (50mg/kg Temozolomide adds 10mg/kg to bind peptide polymer to administering drug combinations group
Micella), low dosage Temozolomide administering drug combinations group (10mg/kg Temozolomide adds 10mg/kg to bind peptide polymer micella), with lotus
The life span of tumor nude mice is metrics evaluation therapeutic effect.
Specific embodiment
It will be helpful to further understand the present invention by following embodiment description, but the invention is not limited to described below
Range.
Embodiment 1
1) target function material is prepared:
Prepare RGD- polyethylene glycol-polylactic acid
Membrane material is realized with reacting for maleimide contained by Mal-PEG3400-PLA2000 by the free sulfhydryl groups of polypeptide
Synthesis.Specific reaction is as follows: 10mg Mal-PEG3400-PLA2000 is dissolved in 1mL methanol, after revolving forms a film, with 1ml pH
Reaction is added several times, reacts 5h at room temperature for 8.0 PB buffer aquation.Sample is collected, (dialysis membrane is cut to pure water dialysis
Staying molecular weight is 3500) to stay overnight, and free polypeptide is removed, up to RGD- polyethylene glycol-polylactic acid after freeze-drying；
Prepare RGD- polyethylene glycol-distearoyl phosphatidyl ethanolamine
Membrane material is realized with reacting for maleimide contained by Mal-PEG3400-DSPE by the free sulfhydryl groups of polypeptide
Synthesis.Specific reaction is as follows: 10mg Mal-PEG3400-DSPE is dissolved in 1mL DMF, and 2mg polypeptide is dissolved in the PBS of 3mL pH7.5
Solution reacts 1h after the two mixing and ultrasonic bubble removing at room temperature.Sample is collected, (dialysis membrane retains molecule to pure water dialysis
3500) amount is overnight, removes free polypeptide, up to RGD- polyethylene glycol-distearoyl phosphatidyl ethanolamine after freeze-drying；
Prepare LyP-1- polyethylene glycol-polylactic acid
Membrane material is realized with reacting for maleimide contained by Mal-PEG3400-PLA2000 by the free sulfhydryl groups of polypeptide
Synthesis.Specific reaction is as follows: 10mg Mal-PEG3400-PLA2000 is dissolved in 1mL methanol, after revolving forms a film, with 1ml pH
The Lyp-1 polypeptides reactive of 1.2 times of moles is added several times, reacts 5h at room temperature for 8.0 PB buffer aquation.Collect sample
Product overnight to pure water dialysis (dialysis retaining molecular weight is 3500) remove free polypeptide, up to the poly- second two of LyP-1- after freeze-drying
Alcohol-polylactic acid；
Prepare LyP-1- polyethylene glycol-distearoyl phosphatidyl ethanolamine
Membrane material is realized with reacting for maleimide contained by Mal-PEG3400-DSPE by the free sulfhydryl groups of polypeptide
Synthesis.Specific reaction is as follows: 10mg Mal-PEG3400-DSPE is dissolved in 1mL DMF, and 2mg polypeptide is dissolved in the PBS of 3mL pH7.5
Solution reacts 1h after the two mixing and ultrasonic bubble removing at room temperature.Sample is collected, (dialysis membrane retains molecule to pure water dialysis
3500) amount is overnight, removes free polypeptide, up to LyP-1- polyethylene glycol-distearoyl phosphatidyl ethanolamine after freeze-drying；
Prepare CDX-polyethylene glycol-polylactic acid
Membrane material is realized with reacting for maleimide contained by Mal-PEG3400-PLA2000 by the free sulfhydryl groups of polypeptide
Synthesis.Specific reaction is as follows: 10mg Mal-PEG3400-PLA2000 is dissolved in 1mL methanol, after revolving forms a film, with 1ml pH
The CDX polypeptides reactive of 1.2 times of moles is added several times, reacts 5h at room temperature for 8.0 PB buffer aquation.Sample is collected,
Overnight to pure water dialysis (dialysis retaining molecular weight is 3500), free polypeptide is removed, up to CDX- polyethylene glycol after freeze-drying
Lactic acid；
Prepare CDX- polyethylene glycol-distearoyl phosphatidyl ethanolamine
Membrane material is realized with reacting for maleimide contained by Mal-PEG3400-DSPE by the free sulfhydryl groups of polypeptide
Synthesis.Specific reaction is as follows: 10mg Mal-PEG3400-DSPE is dissolved in 1mL DMF, and 2mg polypeptide is dissolved in the PBS of 3mL pH7.5
Solution reacts 1h after the two mixing and ultrasonic bubble removing at room temperature.Sample is collected, (dialysis membrane retains molecule to pure water dialysis
3500) amount is overnight, removes free polypeptide, up to CDX- polyethylene glycol-distearoyl phosphatidyl ethanolamine after freeze-drying；
Prepare folic acid-polyethylene glycol-distearoylphosphatidylethanolamine
It is anti-with carboxyl succinimide activated contained by NHS-PEG3400-DSPE by ethylenediamine by after folic acid amination
It should realize the synthesis of membrane material.Specific reaction is as follows: 10mg NHS-PEG3400-DSPE is dissolved in 1mL DMF, and 5mg folic acid is dissolved in
The PBS solution of 3mL pH7.5 reacts 1h after the two mixing and ultrasonic bubble removing at room temperature.Sample is collected, is dialysed to pure water
(dialysis retaining molecular weight is 3500) overnight, removes free folic acid, up to folic acid-polyethylene glycol-distearyl phosphorus after freeze-drying
Acyl ethanol amine；
2) preparation bookbinding peptide sPMI polymer micelle preparation
Weigh the polyethylene glycol-polylactic acid (PEG of the methoxy group of 90% mass ratio2000-PLA2000) and 5% mass ratio
RGD cyclic peptide-polyethylene glycol-polylactic acid (RGD-PEG2000-PLA2000) as the material for preparing polymer micelle.By above-mentioned material
Material and bookbinding peptide sPMI are dissolved in methanol, and wherein the mass ratio of polypeptide and material is 1: 9.It is slow to be placed in constant pressure on Rotary Evaporators
After evaporation film forming, it is placed in vacuum drying phase overnight, removes remaining organic solvent.Add 1mL physiological saline, 37 DEG C of aquations
20min, G50 gel post separation are dissociated polypeptide, and the bookbinding peptide polymer micellar preparation that partial size is about 23nm is obtained.After measured, this is poly-
Closing object micellar preparation is about 99% to the encapsulation rate of bookbinding peptide.
3) the Anticancer Activity in vitro evaluation of peptide polymer micellar preparation is bound
With contain 10% fetal calf serum DMEM culture solution, in carbon dioxide incubator (37 DEG C, 5%CO2, saturated humidity)
Continuous culture, makes human glioma cell U87 be in adhered state.The monolayer cultivation U87 cell of logarithmic growth phase is used
0.25% trypsase and 0.02% disodium ethylene diamine tetraacetate digest and blow and beat into individual cells, and cell is suspended in culture solution
In, it counts, with 3000, every hole cell inoculation in 96 well culture plates, every 200 μ L of pore volume, when bed board reserves not celliferous sky
White culture fluid apertures.Culture is administered afterwards for 24 hours；
Above-mentioned 96 orifice plates for being inoculated with U87 cell are taken, culture solution in hole is discarded, is changed to the culture solution of various concentration gradient
Prepare RGD-PEG-PLA/sPMI micella, empty carrier RGD-PEG-PLA micella, free polypeptide sPMI or positive control Nutlin-3
(20 μ L are added in every hole), each concentration is all provided with three wells (experimental port).It reserves three and the hole of culture solution is only added as control wells.
After cultivating 72h, 20 every L MTT solution (5mg/mL) are added in every hole, after being further cultured for 4h in 37 DEG C of incubators, remove liquid in hole
Body and every hole are added 1505 and move DMSO, measure each hole absorbance value at Detection wavelength 490nm after being incubated at room temperature 15min.It presses
Following formula calculates cell survival rate:
Survival rate maps to drug concentration logarithm, and calculation of half inhibitory concentration (IC50).The results show that RGD-
PEG-PLA/sPMI micella can effectively kill U87 cell, IC50About 10.7 μM, with positive control Nutlin-3 quite (7.6 μ
M), prompt the polymer micelle preparation that there are stronger external anticancer potentiality.
4) anti-cancer activity in vivo for binding peptide polymer micellar preparation evaluates (subcutaneous tumors)
The foundation of U87 subcutaneous tumors animal pattern: the U87 cell of logarithmic growth phase, every nude mouse are inoculated with 6 born of the same parents, often6It is a
Cell (is dispersed in 100 dispersion PBS buffer solution).Cell inoculation is subcutaneous in the right shoulder blade of nude mouse.Every two after inoculation
Major diameter (the D of its tumor of measurementmax) and minor axis (Dmin), knurl product (V) is calculated according to the following formula:
V=[Dmax×(Dmin)2]/2
For kind after tumor 14 days, nude mouse subcutaneous tumors size reaches 50~120mm3When, start to carry out effect experiment.It is randomly divided into
6 groups, every group of 8 U87 subcutaneous tumors model nude mouses；
Experimental group and dosage regimen: physiological saline group, polypeptide sPMI group of dissociating contain the RGD-PEG- of bookbinding peptide sPMI
PLA polymer micelle low dosage (4mg/kg) and high dose group (10mg/kg), Temozolomide group (50mg/kg takes orally) and joint
Administration group (50mg/kg Temozolomide+10mg/kg bind peptide polymer micella), in the 1st day, the 3rd day, the 5th day, the 7th day and the
It is administered within 9 days；
Effect experiment carries out 14 days altogether, and the major diameter and minor axis of tumor are measured before first administration, obtains knurl product (V0), later every two
It equally operates to obtain knurl product (Vd), by Rd=Vd/V0Knurl product relative value (R is calculatedd), using the time as abscissa, with Rd
For ordinate mapping, obtains knurl product relative value and change over time curve evaluation therapeutic effect；
Knurl product relative value changes over time the results show that comparing physiological saline group, and free polypeptide sPMI group can slightly press down
The growth (P < 0.01) of U87 subcutaneous tumors processed contains the RGD-PEG-PLA polymer micelle low dosage (4mg/kg) of bookbinding peptide sPMI
The growth of U87 subcutaneous tumors can be significantly inhibited with high dose group (10mg/kg), and concentration dependent is presented, prompts the polymer latex
Beam preparation has stronger antitumor potentiality (P < 0.001) in vivo.Compared to independent Temozolomide group, peptide sPMI polymer latex is bound
After beam and Temozolomide are administered in combination, tumor killing effect (P < 0.001) can be further enhanced.
5) anti-cancer activity in vivo for binding peptide polymer micella delivery system evaluates (brain primary tumor)
The foundation of U87 brain primary tumor animal pattern: the U87 cell of logarithmic growth phase, every nude mouse inoculation 5 × 105
A cell (being scattered in 5 μ L PBS buffer solution).After nude mouse anesthesia, fixed with stereotaxic apparatus, cell inoculation is in brain line
Shape body right part (0.6mm, side 1.8mm, deep 3mm before bregma)；
It is administered the 10th day after inoculated tumour cell.Lotus knurl nude mouse is randomly divided into 5 groups, every group 10；
Experimental group and dosage regimen are as follows: physiological saline group, contain the RGD-PEG-PLA polymer latex of bookbinding peptide sPMI
Beam group (10mg/kg), Temozolomide group (50mg/kg takes orally), (50mg/kg Temozolomide+10mg/kg bookbinding of administering drug combinations group
Peptide polymer micella), (10mg/kg Temozolomide+10mg/kg binds peptide polymer glue to low dosage Temozolomide administering drug combinations group
Beam), it is administered within the 10th day, the 12nd day, the 14th day, the 16th day and the 18th day after kind of tumor.Draw each group Kaplan-Meier existence
Curve evaluates therapeutic effect；The results show that containing the RGD-PEG-PLA polymer of bookbinding peptide sPMI compared with physiological saline group
Micella group (10mg/kg) has the life span of certain lengthening model animal.(middle position is raw compared with high dose Temozolomide
Depositing the time is 59 days), administering drug combinations group (50mg/kg Temozolomide+10mg/kg binds peptide polymer micella) can significantly prolong
The median survival time (74 days) of long animal pattern；With low dosage Temozolomide (10mg/kg) and bookbinding peptide polymer micella
After (10mg/kg) is administered in combination, median survival time (55 days) is close with independent high dose Temozolomide group (59 days), as a result
Show that bookbinding peptide polymer micellar preparation of the invention has stronger anti-brain tumor potentiality in vivo, and can be obviously improved for not
The anti-brain tumor drug effect of azoles amine.
Claims (5)
1. a kind of bookbinding peptide polymer micellar preparation for anticancer, which is characterized in that by target function material, amphipathic high score
Sub- block copolymer and bookbinding peptide composition, in which: target function material as one component part of polymer micelle for identification
Target cell, amphipathy macromolecule block copolymer is as polymer micelle skeleton part for containing and delivering bookbinding peptide；
The target function material is the targeted molecular for specifically binding target cell, with amphipathy macromolecule block copolymerization
The active targeting compound of object coupling；
The amphipathy macromolecule block copolymer is selected from: polyethylene glycol-polylactic acid, polyethylene glycol-lactic-glycolic acid are total
Polymers, polyethylene glycol-distearoyl phosphatidyl ethanolamine or polyethylene glycol-dipalmitoylphosphatidylcholine；
The bookbinding peptide is selected to carry out the polypeptide that mirror image display technique of bacteriophage screens for cancer protein MDM2
PMI polypeptide, PMI-N8A peptide, sequence TSFAEYWN-LLSP, TSFAEYWALLSP or TSFAEYWALLS, setting site is
4,11；
The polymer micelle preparation partial size is 30-200nm.
2. the bookbinding peptide polymer micellar preparation according to claim 1 for anticancer, which is characterized in that described is amphipathic
Its end group of polyethylene glycol in high-molecular block copolymer is active group, is selected from dimaleoyl imino, sulfydryl, amide groups, ammonia
One of base, carboxyl, biotin or Avidin.
3. the bookbinding peptide polymer micellar preparation according to claim 1 for anticancer, which is characterized in that the specificity
Targeted molecular in conjunction with target cell is smaller ligand folic acid or oxidation ascorbic acid.
4. the bookbinding peptide polymer micellar preparation according to claim 1 for anticancer, which is characterized in that specific binding target
The targeted molecular of cell is polypeptide ligand, is selected from RGD peptide, LyP-1 peptide or CDX peptide.
5. the bookbinding peptide polymer micellar preparation according to claim 1 for anticancer, which is characterized in that the bookbinding peptide
The combination of competitive antagonism MDM2/MDMX and cancer suppressor protein p53 restore p53 protein active.
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| WO2019165576A1 (en) * | 2018-02-27 | 2019-09-06 | President And Fellows Of Harvard College | Methods for treating cancer and protecting renewable tissues |
| CN109731104A (en) * | 2018-12-29 | 2019-05-10 | 苏州明基医院有限公司 | A kind of polypeptide-rare earth material delivery system and preparation method and application |
| CN111909241B (en) * | 2020-07-09 | 2021-12-17 | 西安交通大学医学院第一附属医院 | Polypeptide nano hybrid and preparation method and application thereof |
| CN113845598B (en) * | 2021-09-18 | 2023-06-30 | 中国人民解放军海军军医大学 | Protein targeting chimera degradation MDM2/MDMX protein staple peptide conjugate and application thereof |
| CN114452405A (en) * | 2022-02-28 | 2022-05-10 | 西安交通大学医学院第一附属医院 | Metal organic supermolecule with functions of inducing iron death and activating p53 |
| CN115073554B (en) * | 2022-06-27 | 2024-04-19 | 上海交通大学医学院附属瑞金医院 | Staple peptide and application thereof in preparation of medicines for treating pancreatic cancer |
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