CN103936829A - Polypeptide, targeted drug carrier, preparation method of targeted drug carrier, pharmaceutical composition, and preparation method of pharmaceutical composition - Google Patents
Polypeptide, targeted drug carrier, preparation method of targeted drug carrier, pharmaceutical composition, and preparation method of pharmaceutical composition Download PDFInfo
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Abstract
The invention discloses a polypeptide, which contains an amino acid sequence represented by the SEQ ID No.1. The invention also discloses a targeted drug carrier and a preparation method thereof. The preparation method comprises the following step: contacting the polypeptide provided by the invention with a drug carrier containing a group represented by the formula (1) so as to form a covalent linkage, which is represented by the formula (2), between the polypeptide and the drug carrier. The invention also discloses a pharmaceutical composition and a preparation method thereof. The preparation method comprises the following steps: (1) mixing a hydrophobic drug with a substance containing the targeted drug carrier mentioned above in an organic solvent, drying to remove the organic solvent so as to obtain a dried material; (2) contacting the dried material with a buffer solution. The polypeptide, targeted drug carrier, and pharmaceutical composition all have a high efficient targeting property and a very strong tumor penetrating performance.
Description
Technical field
The present invention relates to pharmaceutical chemistry field, be specifically related to a peptide species, a kind of target medicine carrier and preparation method thereof and a kind of pharmaceutical composition and preparation method thereof.
Background technology
Many antitumor drugs are due to a little less than the penetrating power of tumor tissues position, and under high dosage, normal cell toxic side effect have been limited to its result for the treatment of.Conventionally in noumenal tumour, medicine can only be penetrated near the region of 3-5 the cell dia of tumor vessel, is difficult to overcome the interstitial that inside tumor is higher and presses, and then be penetrated in tumor epithelial cell, finally causes drug effect to reduce and even makes body produce resistance.At present, the nano-carrier energy carrier band medicine that many targeted moleculars (as target polypeptide RGD, Transferrins,iron complexes, folic acid etc.) are modified accumulates at tumor locus, but still only has a small amount of medicine to enter tumor tissues and to be penetrated into away from tumor vascular tumor epithelial cell through vascular endothelial cell.Therefore, how to make medicine penetration rate of blood tube wall be penetrated into the main challenge that has become oncotherapy in tumor epithelial cell effectively, need the efficient target tumor of exploitation badly and effectively at the targeted molecular of tumor locus infiltration.
Research discovery, polypeptide primitive R/K × × R/K can carry medicine infiltration in cell or tissue, and this is mainly by neural pilin-1 receptors bind of R/K × × R/K and tumor vessel inwall or tumor cell surface high expression level mediation.And R/K × × R/K just has activity while being only present in peptide sequence C-terminal position, is referred to as CendR.Interaction between CendR primitive and neural pilin-1 is the crucial determinative of effectively permeating.
Therefore the targeted molecular of, needing a kind of efficiently target tumor that contains this polypeptide primitive of exploitation badly and effectively permeating at tumor locus.
Summary of the invention
The object of the invention is target tumor efficiency in order to overcome existing targeted molecular low, in the low shortcoming of tumor locus rate of permeation, provide one to there is efficient target and infiltrative polypeptide, and a kind of target medicine carrier and preparation method thereof, a kind of pharmaceutical composition and preparation method thereof.
Based on above discovery, the invention provides a peptide species, wherein, this polypeptide has the aminoacid sequence as shown in SEQ IDNo:1.
On the other hand, the invention provides a kind of preparation method of target medicine carrier, wherein, the method comprises polypeptide provided by the invention contacted with containing the pharmaceutical carrier of group as the formula (1), makes to form between described polypeptide and described pharmaceutical carrier as the formula (2) covalently bound.
On the one hand, the present invention also provides a kind of preparation method of pharmaceutical composition again, and wherein, the method comprises:
(1) in organic solvent, hydrophobic drug is mixed with the material that contains target medicine carrier provided by the invention, be then dried to remove described organic solvent, obtain dried material;
(2) described dried material is contacted with buffered soln.
Polypeptide provided by the invention, target medicine carrier and pharmaceutical composition had both had efficient targeting and had also had stronger tumour penetrating power.
Other features and advantages of the present invention are described in detail the embodiment part subsequently.
Brief description of the drawings
Accompanying drawing is to be used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the transmission electron microscope picture of the pharmaceutical composition prepared of embodiment 1 and comparative example 1.
Fig. 2 is the size distribution figure of the pharmaceutical composition prepared of embodiment 1 and comparative example 1.
Fig. 3 is the potential image of the pharmaceutical composition prepared of embodiment 1 and comparative example 1.
Fig. 4 is the drug release graphic representation of the pharmaceutical composition prepared of embodiment 1 and comparative example 1.
Fig. 5 is the cytotoxicity experiment result figure of the pharmaceutical composition prepared of embodiment 1 and comparative example 1.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is elaborated.Should be appreciated that embodiment described herein, only for description and interpretation the present invention, is not limited to the present invention.
First aspect, the invention provides a peptide species, and wherein, this polypeptide has the aminoacid sequence shown in SEQ ID No:1.
The present inventor's discovery, peptide sequence CRGDK can initiatively be targeted to tumor vascular endothelial cell or the tumor cell surface of neural pilin-1 of high expression level, and then plays a role.
Second aspect, the invention provides a kind of preparation method of target medicine carrier, wherein, the method comprises polypeptide provided by the invention contacted with containing the pharmaceutical carrier of group as the formula (1), makes to form between described polypeptide and described pharmaceutical carrier as the formula (2) covalently bound.
According to the present invention, the C-terminal (Lys end) of described peptide sequence need be exposed to outside and could combine with neural pilin-1 of tumor cell surface specifically, therefore, the method of preparing target medicine carrier provided by the invention comprises: the group as the formula (1) that the Cys amino-terminal end of described polypeptide and pharmaceutical carrier end are contained, be maleoyl amido, form as the formula (2) covalently bound.
According to the present invention, described polypeptide with described in contain maleoyl amido pharmaceutical carrier in the consumption of maleoyl amido have no particular limits, as long as described polypeptide and the maleoyl amido in the pharmaceutical carrier that contains maleoyl amido can form described above covalently bound, in preferred situation, described polypeptide with described in contain maleoyl amido pharmaceutical carrier in the mol ratio of maleoyl amido be 1-5:1.
According to the present invention, described covalently bound condition can be selected in wide range, as long as ensure to form described above covalently bound.Described covalently bound condition can be: pH value is 6-7, and temperature is 20-40 DEG C, and the time is 40-60 hour.
According to the present invention, described covalently bound reaction is carried out in solution, described solution preferred buffer, and described damping fluid can be the various damping fluid for the preparation of pharmaceutical carrier well known in the art, for example, can be hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid of 40-60mM.Wherein, the polypeptide adding in described solution and the amount of pharmaceutical carrier have no particular limits, as long as ensure the preferred proportion of above-mentioned two kinds of materials, preferably, with respect to solution every milliliter described, the total amount of the interpolation of above-mentioned two kinds of materials can be 5-10mg.
In the present invention, have no particular limits for the selection of the described pharmaceutical carrier that contains maleoyl amido.The pharmaceutical carrier that for example, contains maleoyl amido, can be the amphiphilic diblock copolymer that contains maleoyl amido and/or the amphiphilic dendrimer that contains maleoyl amido.Wherein, the amphiphilic diblock copolymer that contains maleoyl amido described in can be the DSPE-PEG 2000(DSPE-PEG that contains maleoyl amido
2000-MAL); The described amphiphilic dendrimer that contains maleoyl amido can be the polyamide-amide dendrimer that contains maleoyl amido.
According to the present invention, the method also comprises removes the polypeptide not connecting in the solution after covalently bound, and the solution of removing after polypeptide is dried to the dry powder that obtains this target medicine carrier.Describedly remove polypeptide and dry method is conventionally known to one of skill in the art, for example, described in remove polypeptide method can be dialysis, described dry method can be lyophilize.
Described dialysis refers to the described liquid completing after described contact is placed in to the dialysis capsule with dialysis membrane isolation, and completes the dialysis capsule submergence of the liquid after described contact or be partially submerged in dialyzate described in making to contain.Described dialysis membrane can be can molecular weight cut-off 2000 above materials dialysis membrane, for example, purchased from upper sea base star bio tech ltd, article No. is the product of MD45 (2000).Described dialyzate refers to that osmotic pressure for example, lower than the described liquid completing after described contact, deionized water.The concrete grammar of dialysis is conventionally known to one of skill in the art, does not repeat them here.
The third aspect, provides the target medicine carrier of being prepared by preparation method provided by the invention.
Fourth aspect, the invention provides a kind of preparation method of pharmaceutical composition, and wherein, the method comprises:
(1) in organic solvent, hydrophobic drug is mixed with the material that contains target medicine carrier provided by the invention, be then dried to remove described organic solvent, obtain dried material;
(2) described dried material is contacted with buffered soln.
According to the present invention, in step (1), described in contain target medicine carrier provided by the invention material in the content of target medicine carrier can in very large range change, for example, can be 20-100 % by weight.Wherein, the material in described material except target medicine carrier of the present invention can be, for example common pharmaceutical carrier does not connect the pharmaceutical carrier of maleoyl amido, for example, and DSPE-PEG 2000(DSPE-PEG
2000).
The consumption of described organic solvent, the material that contains target medicine carrier and hydrophobic drug has no particular limits.Considering cost and efficiency, preferably, with respect to the described hydrophobic drug of every weight part, the consumption of described hybrid medicine carrier is 3-10 weight part; The consumption of described organic solvent is 1-4 parts by volume.
In the present invention, described hydrophobic drug can be the known various hydrophobic drugs of field of medicaments personnel, for example, can be at least one in Zorubicin, taxol and docetaxel.Wherein, hydrophobicity Zorubicin refer to by commercially available doxorubicin hydrochloride and after obtain Zorubicin, hydrophobicity Zorubicin also can be by commercially available.
Wherein, the kind of organic solution has no particular limits, and for example, can be at least one in chloroform, methyl alcohol and ethanol, is preferably by the chloroform of the volume ratio mixing of 2-4:1 and the mixed organic solvents of methyl alcohol.
Described dry method is conventionally known to one of skill in the art, and for example, the method that can revolve steaming with reference to disclosed employing vacuum in pharmaceutics (People's Health Publisher, 2007 publish) is carried out.
According to the present invention, in step (2), the various damping fluids of what the damping fluid that described and dried material contacts can be well known to those skilled in the art can be used for pharmaceutical composition, for example, can be phosphoric acid salt (PBS) damping fluid, HEPES damping fluid or and the physiological saline of pH7.3-7.5.Be preferably the phosphate buffered saline buffer that pH value is 7.3-7.5.Wherein, described pH value for the phosphate buffered saline buffer of 7.3-7.5 be the aqueous solution of Repone K, the Sodium phosphate dibasic of 2.8-3.0g/L and the potassium primary phosphate of 0.15-0.25g/L of the sodium-chlor that contains 7.5-8.5g/L, 0.15-0.25g/L.
Wherein, in step (1), dried material, in the time contacting with damping fluid, can form targeted nano micella.Wherein, the condition of described contact has no particular limits, as long as it can form nano-micelle in damping fluid, the condition of described contact can comprise: temperature is 40-60 DEG C, and the time is 20-120min.
Wherein, the consumption of described damping fluid also has no particular limits, as long as can change described dried thing and hydrophobic drug into nano-micelle, preferably, with respect to dried material described in 1mg, the add-on of described damping fluid is 0.1-0.5mL.
The pharmaceutical composition that the present invention also provides preparation method as above to prepare.Typically, the micella that is arranged in the phosphate buffered saline buffer that pH value is 7.3-7.5 obtaining can be used as pharmaceutical composition according to the present invention, in order to ensure that described dried material is fully converted into nano-micelle, the method also comprises and leaves standstill 1-3 hour by contacting with damping fluid at mixed solution after finishing and 20-40 DEG C.
According to the present invention, the method also comprises free hydrophobic drug in the mixed solution after leaving standstill separated, and the method for described separation is that those skilled in the art are known, for example, can use the aseptic filter membrane of 0.22 μ m to filter.
Wherein, described nano-micelle is the amphipathic nature block polymer thermodynamically stable colloidal solution of one that self-assembly forms in water.The particle diameter of described nano-micelle is 10-50nm.The particle diameter of described micella refers to the numerical value of the dynamic light scattering determination in reference literature (nanomaterial science, press of Harbin Engineering University, version in 2002).
The 5th aspect, the present invention also provides the pharmaceutical composition of preparing according to preparation method as above.
Below will describe the present invention by embodiment.In following examples, polypeptide is synthesized by gill biochemical corp; DSPE-PEG
2000-MAL and DSPE-PEG
2000all, purchased from avanti company, article No. is respectively 880126P and 880120P.Hydrophobicity Zorubicin is purchased from LC Laboratories, and trade names are D-4099; Hydrophobicity docetaxel and hydrophobicity taxol are all purchased from Beijing Hua Fenglianbo Science and Technology Ltd..Transmission electron microscope picture is observed and is obtained by the transmission electron microscope (Tecnai G220S-TWIN, 200kV) of FEI Co.; Particle size analysis collection of illustrative plates is measured by the laser particle analyzer dynamic light scattering (Zetasizer NanoZS) of Malvern company.Cell viability is measured by the multi-functional microplate reader of continuous spectrum (Tecaninfinite M200) and is detected.Dialysis tubing is purchased from upper sea base star bio tech ltd, and article No. is MD45 (2000).People source mammary cancer high invasion and attack sexual cell MDA-MB-231 and people source breast cancer cell MCF-7 are all purchased from American Type Culture Collection (ATCC).
Embodiment 1
The present embodiment is used for illustrating polypeptide provided by the invention, target medicine carrier and pharmaceutical composition and preparation method thereof
(1) take respectively the polypeptide of 3 milligrams and the DSPE-PEG of 15 milligrams
2000the mol ratio of the maleoyl amido in-MAL(polypeptide and described pharmaceutical carrier is 1:1) and be dissolved in hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid (pH6.5) that 2mL concentration is 50mM, at 30 DEG C, contact 48 hours, in carrying, to stay molecular weight be to dialyse to remove the polypeptide not connecting in 2000 dialysis tubing to the solution of contact after finishing, after dialysis finishes and carry out lyophilize and obtain the dry powder that contains target medicine carrier of the present invention.
(2) in eggplant-shape bottle by the dry powder that contains target medicine carrier preparing in 2mg step (1), 8mg common drug carrier DSPE-PEG
2000be dissolved in the chloroform of 4mL and mixing solutions that methanol solution volume ratio is 3:1 with the hydrophobicity Zorubicin of 2mg, and carry out rotary evaporation in vacuo with reference to disclosed method in pharmaceutics (People's Health Publisher publishes for 2007), obtain the dried material of 12mg.
(3) in the eggplant-shape bottle that contains dried material in step (2), add the PBS damping fluid 2mL of the pH7.4 of 60 DEG C to contact, the condition of contact is: temperature is 60 DEG C, and the time is 30min.Solution after contact is finished leaves standstill 2 hours in 25 DEG C, finally the solution obtaining is filtered to the aseptic filter membrane of 0.22 μ m, removes free Zorubicin, obtains the pharmaceutical composition of micellization.
Embodiment 2
The present embodiment is used for illustrating polypeptide provided by the invention, target medicine carrier and pharmaceutical composition and preparation method thereof
(1) take respectively the polypeptide of 15 milligrams and the DSPE-PEG of 15 milligrams
2000the mol ratio of the maleoyl amido in-MAL(polypeptide and described pharmaceutical carrier is 5:1) and be dissolved in hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid (pH6.0) that 3mL concentration is 40mM, at 20 DEG C, contact 60 hours, in carrying, to stay molecular weight be to dialyse to remove the polypeptide not connecting in 2000 dialysis membrane to the solution of contact after finishing, after dialysis finishes and carry out lyophilize and obtain the dry powder that contains target medicine carrier of the present invention.
(2) in eggplant-shape bottle, the docetaxel of the dry powder that contains target medicine carrier preparing in 10mg step (1) and 1mg is dissolved in the chloroform of 3mL and mixing solutions that methanol solution is 2:1, and with reference to pharmaceutics (People's Health Publisher, 2007 publish) in disclosed method carry out rotary evaporation in vacuo, obtain the dried material of 11mg.
(3) in the eggplant-shape bottle that contains dried material in step (2), add the PBS damping fluid 1.1mL of the pH7.4 of 60 DEG C to contact, the condition of contact is: temperature is 40 DEG C, and the time is 120min.Solution after contact is finished leaves standstill 1 hour in 40 DEG C, finally the solution obtaining is filtered to the aseptic filter membrane of 0.22 μ m, removes free docetaxel, obtains the pharmaceutical composition of micellization.
Embodiment 3
The present embodiment is used for illustrating polypeptide provided by the invention, target medicine carrier and pharmaceutical composition and preparation method thereof
(1) take respectively the polypeptide of 6 milligrams and the DSPE-PEG of 15 milligrams
2000the mol ratio of the maleoyl amido in-MAL(polypeptide and described pharmaceutical carrier is 2:1) and be dissolved in hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid (pH7) that 4mL concentration is 60mM, at 40 DEG C, contact 40 hours, in carrying, to stay molecular weight be to dialyse to remove the polypeptide not connecting in 2000 dialysis membrane to the solution of contact after finishing, after dialysis finishes and carry out lyophilize and obtain the dry powder that contains target medicine carrier of the present invention.
(2) in eggplant-shape bottle by the dry powder that contains target medicine carrier preparing in 2mg step (1), 10mg common drug carrier DSPE-PEG
2000be dissolved in the chloroform of 4mL and mixing solutions that methanol solution is 4:1 with the taxol of 4mg, and carry out rotary evaporation in vacuo with reference to disclosed method in pharmaceutics (People's Health Publisher publishes for 2007), obtain the dried material of 16mg.
(3) in the eggplant-shape bottle that contains dried material in step (2), add the HEPES damping fluid 8mL of the pH7.4 of 60 DEG C to contact, the condition of contact is: temperature is 50 DEG C, and the time is 20min.Solution after contact is finished leaves standstill 3 hours in 20 DEG C, finally the solution obtaining is filtered to the aseptic filter membrane of 0.22 μ m, removes free taxol, obtains the pharmaceutical composition of micellization.
Embodiment 4
The present embodiment is used for illustrating polypeptide provided by the invention, target medicine carrier and pharmaceutical composition and preparation method thereof
Carry out synthetic, the preparation of target medicine carrier and the preparation of pharmaceutical composition of polypeptide according to the method for embodiment 1, wherein, different, in step (3), the condition of contact is 30 DEG C, and the time is 30min.
Comparative example 1
The present embodiment is for illustrating not containing the pharmaceutical carrier of polypeptide of the present invention and the preparation of pharmaceutical composition
Carry out the preparation of pharmaceutical carrier and the preparation of pharmaceutical composition according to the method for embodiment 1, wherein, different, in step (1), do not add polypeptide of the present invention.
Test implementation example 1-4
(1) mensuration of pharmaceutical composition form and particle diameter
Use transmission electron microscope according to the method in its specification sheets, observe the pharmaceutical composition of preparing in embodiment 1-4.Use laser particle analyzer according to the method in its specification sheets, the pharmaceutical composition of preparing in embodiment 1-4 is carried out respectively to Dynamic Light Scattering Determination, and the median size that records the pharmaceutical composition of preparing in embodiment 1-4 is respectively 13.10nm, 11.05nm, 12.56nm and 15.06nm.Wherein, as shown in Figure 1, size distribution as shown in Figure 2 for the transmission electron microscope picture of the pharmaceutical composition of preparing in embodiment 1.
(2) potentiometric analysis of pharmaceutical composition
Use laser particle analyzer according to the method in its specification sheets, the pharmaceutical composition of preparing in embodiment 1-4 is carried out to Zeta-potentiometric analysis, record be respectively-6.92mV of pharmaceutical composition the mean charge ,-8.31mV ,-the 6.03mV ,-5.34mV that in embodiment 1-4, prepare, show that it is with faint negative charge.Wherein, in embodiment 1, the charge distribution of the pharmaceutical composition of preparation is shown in Fig. 3.
(3) release characteristics of pharmaceutical composition Chinese traditional medicine
Respectively the pharmaceutical composition of preparing in 0.2ml embodiment 1-4 is placed in to 4 different dialysis tubings, with the PBS phosphate buffered saline buffer (aqueous solution of the sodium-chlor that contains 8.0g/L, the Repone K of 0.2g/L, the disodium hydrogen phosphate dodecahydrate of 2.9g/L and the potassium primary phosphate of 0.2g/L of the pH7.4 of 30ml, pH7.4) be release medium, as neutral group; Respectively the pharmaceutical composition of preparing in 0.2ml embodiment 1-4 is placed in to 4 different dialysis tubings, with the pH5.0 phosphate buffered saline buffer (aqueous solution of the sodium-chlor that contains 8.0g/L, the Repone K of 0.2g/L, the disodium hydrogen phosphate dodecahydrate of 2.9g/L and the potassium primary phosphate of 0.2g/L of 30ml, pH5.0) be release medium, as acid group.At 37 DEG C, put into release medium from dialysis tubing and be made as for 0 moment that quarter, 0.5,1,2,4,6,9,12,24 hours time, draw respectively the outer liquid of dialysis tubing of the neutral group of 1ml and acid group, and supplement release medium separately simultaneously, cumulative volume is remained unchanged.Adopt ultraviolet spectrophotometer to measure the concentration of each time point extracellular fluid dialysis Chinese traditional medicine of neutral group and acid group, according to the method in document (Biopharmaceutics and Pharmacokinetics, People's Health Publisher publish for 2007), calculate release amount of medicine.Result shows, the pharmaceutical composition of preparing in embodiment 1-4 can discharge rapidly under the condition of pH5.0, release rate and burst size, far away higher than the release under the condition of pH7.4, illustrate that pharmaceutical composition provided by the invention has the drug release characteristics of pH sensitivity.Wherein, in embodiment 1, the pharmaceutical composition of preparation release conditions is in time shown in Fig. 4, when 24h, its burst size under the condition of pH7.4 is 33.8%, and burst size under the condition of pH5.0 is 77.8%, wherein, the per-cent of the medicine total amount that described burst size adds for discharged medicine accounts for, its release rate is faster under acidic conditions as seen.
(4) cell toxicity test
Select people source mammary cancer high invasion and attack sexual cell MDA-MB-231 cell and people source MCF-7 Breast Cancer Cell to detect the experiment of the targeting of the pharmaceutical composition of preparing in embodiment 1-4.Wherein, neural pilin-1 of the high invasion and attack of people source mammary cancer sexual cell MDA-MB-231 cell surface high expression level acceptor, and neural pilin-1 of low expression, people source MCF-7 Breast Cancer Cell surface acceptor.According to document (cell cultures, Si Tuzhenqiang, world book publishing company, 1996) in method above-mentioned two kinds of cells are cultivated, the pharmaceutical composition that then adds respectively embodiment 1-4 to prepare, dosing after 24 hours according to document (cell cultures, Si Tuzhenqiang, world book publishing company, 1996) in method (mtt assay) detect cell survival rate (positive control be the free drug group containing same concentrations hydrophobic drug, negative control is the blank substratum that does not contain hydrophobic drug, wherein calculate by 100% with the survival rate of cell in negative control group).Result show, the pharmaceutical composition in embodiment 1-4 to the fragmentation effect of MDA-MB-231 cell and MCF-7 cell all apparently higher than positive controls.Pharmaceutical composition in embodiment 1 and positive control respectively as shown in Fig. 5-1 and Fig. 5-2, add the concentration of medicine with the densitometer of Zorubicin to the situation of killing and wounding of MDA-MB-231 cell and MCF-7 cell, are respectively 0.01,0.1,1,10 and 100 μ g/ml.Its survival of half for MDA-MB-231 cell concentration is 0.38 μ g/ml, far below the half survival concentration 4.89 μ g/ml of free Zorubicin group; Its survival of half for MCF-7 cell concentration is 1.94 μ g/ml, far below the half survival concentration 6.90 μ g/ml of free Zorubicin group.Pharmaceutical composition prepared by embodiment 4 is 0.94 μ g/ml for the half survival concentration of MDA-MB-231 cell, lower than the half survival concentration 4.89 μ g/ml of free Zorubicin group; Its survival of half for MCF-7 cell concentration is 1.97 μ g/ml, lower than the half survival concentration 6.90 μ g/ml of free Zorubicin group.
Test comparison example 1
According to the method in test case 1-4, pharmaceutical composition is carried out the test of each performance, different, described medicine is the pharmaceutical composition in comparative example 1.Transmission electron microscope picture as shown in Figure 1; As shown in Figure 2, particle diameter is 11.53nm to size distribution; Potentiometric analysis demonstration, mean charge is 0.15mV, charge distribution is shown in Fig. 3; The release characteristics of medicine is measured and shown, under the condition of pH5.0, medicine can discharge rapidly, and release rate and burst size are far away higher than the release under pH7.4 condition, and release conditions is in time seen Fig. 4; Cytotoxic test is shown, pharmaceutical composition prepared by comparative example 1 is 2.08 μ g/ml to the half survival concentration of MDA-MB-231 cell; Half survival concentration to MCF-7 cell is 1.52 μ g/ml.
As can be seen from the above results, pharmaceutical composition provided by the invention with the particle form of pharmaceutical composition that does not carry polypeptide of the present invention all compared with homogeneous, charge distribution is close, and target polypeptide is modified in this explanation explanation can not affect form and the surface potential of the nano particle of formation.Meanwhile, modify target polypeptide and also can not affect the release characteristics to medicine.Cytotoxic result shows, is directed to MDA-MB-231 cell, and the pharmaceutical composition in embodiment 1-4 is to the half survival concentration of cell all lower than the pharmaceutical composition in comparative example 1, and this shows that it has efficient targeting to MDA-MB-231 cell; And be directed to MCF-7 cell, neural pilin-1 of the low expression of cell surface acceptor, so the fragmentation effect to cell and the comparative example of embodiment 1-4 are close, there was no significant difference, therefore, pharmaceutical composition provided by the invention has the efficient targeting for the tumour cell of neural pilin-1 of cell surface high expression level, and can enter efficiently tumour cell and tumour cell is killed, and pharmaceutical composition in comparative example 1 is without this characteristic.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, also can carry out arbitrary combination between various embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. a peptide species, is characterized in that, this polypeptide has the aminoacid sequence as shown in SEQ ID No:1.
2. the preparation method of a target medicine carrier, it is characterized in that, the method comprises polypeptide claimed in claim 1 contacted with containing the pharmaceutical carrier of group as the formula (1), makes to form between described polypeptide and described pharmaceutical carrier as the formula (2) covalently bound.
3. preparation method according to claim 2, wherein, shown in described polypeptide and described pharmaceutical carrier Chinese style (1), the mol ratio of group is 1-5:1.
4. according to the preparation method described in claim 2 or 3, wherein, the condition of described contact comprises: pH value is 6-7, and temperature is 20-40 DEG C, and the time is 40-60 hour.
5. according to the preparation method described in any one in claim 2-4, wherein, described pharmaceutical carrier is the amphiphilic diblock copolymer that contains group as the formula (1) and/or contains the amphiphilic dendrimer of group as the formula (1); The described amphiphilic diblock copolymer of group that contains as the formula (1) comprises and contains the DSPE-PEG 2000 of group as the formula (1); The described amphiphilic dendrimer of group that contains as the formula (1) comprises and contains the polyamide-amide dendrimer of group as the formula (1).
6. the target medicine carrier of being prepared by the preparation method described in any one in claim 1-5.
7. a preparation method for pharmaceutical composition, is characterized in that, the method comprises:
(1) in organic solvent, hydrophobic drug is mixed with the material that contains target medicine carrier claimed in claim 6, be then dried to remove described organic solvent, obtain dried material;
(2) described dried material is contacted with buffered soln.
8. preparation method according to claim 7, wherein, in step (1), with respect to the described hydrophobic drug of every weight part, described in contain target medicine carrier the consumption of material be 3-10 weight part; The consumption of described organic solvent is 1-4 parts by volume.
9. according to the preparation method described in claim 7 or 8, wherein, described hydrophobic drug is selected from least one in Zorubicin, daunorubicin, taxol and docetaxel; Described organic solvent is at least one in chloroform, methyl alcohol and ethanol; Described buffered soln is any one in phosphate buffered saline buffer, hydroxyethyl piperazine second thiosulfonic acid damping fluid and physiological saline.
10. the pharmaceutical composition of being prepared by the method described in any one in claim 1-9.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107334735A (en) * | 2017-08-17 | 2017-11-10 | 国家纳米科学中心 | A kind of eye targeted drug delivery system, eye targeted medicament composition and its preparation method and application |
| CN110812492A (en) * | 2019-11-18 | 2020-02-21 | 内蒙古医科大学 | Small-particle-size drug-loaded micelle of active targeting α v β 3 integrin receptor tumor cells and preparation method thereof |
| CN110812491A (en) * | 2019-11-18 | 2020-02-21 | 内蒙古医科大学 | Small-particle-size quercetin nano-drug targeting α v β 3 integrin receptor high-expression tumor cells and preparation method thereof |
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| CN102397256A (en) * | 2010-09-13 | 2012-04-04 | 复旦大学 | Hydrophilic drug and hydrophobic drug co-loaded target composite nano-drug preparation and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107334735A (en) * | 2017-08-17 | 2017-11-10 | 国家纳米科学中心 | A kind of eye targeted drug delivery system, eye targeted medicament composition and its preparation method and application |
| CN107334735B (en) * | 2017-08-17 | 2020-08-11 | 国家纳米科学中心 | Eye-targeted drug delivery system, eye-targeted drug composition, and preparation method and application thereof |
| CN110812492A (en) * | 2019-11-18 | 2020-02-21 | 内蒙古医科大学 | Small-particle-size drug-loaded micelle of active targeting α v β 3 integrin receptor tumor cells and preparation method thereof |
| CN110812491A (en) * | 2019-11-18 | 2020-02-21 | 内蒙古医科大学 | Small-particle-size quercetin nano-drug targeting α v β 3 integrin receptor high-expression tumor cells and preparation method thereof |
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