CN107551277A - The sensitive targeting phosphatide polyhistidyl nanoparticles of pH for containing antineoplastic - Google Patents

Abstract

The present invention relates to a kind of sensitive targeting phosphatide polyhistidyl nanoparticles of pH for being used to contain cancer therapy drug.The quality percentage composition of its raw material：Polyhistidyl 50~80%；Phosphatide (including phosphatide PEG) 20%~50%；Wherein, phosphatide PEG accounts for the 1%~100% of phosphatide gross mass.Hydrophobic inner core is formed by polyhistidyl, surface modification has polyethylene glycol and cancer target peptide.The phospholipid surface of PEGylation have bio-compatible is good, stability is high, in vivo macrocyclic feature；Histidine kernel can contain hydrophobic anticancer drug in neutral conditions, protonated in tumor microenvironment, mediation carrier occurs current potential and is changed into from negative electricity close to neutrality, carrier is promoted to absorb into born of the same parents, lysosome escape, rapid delivery of pharmaceuticals, effective killing tumor cell are mediated in lysosome after entering born of the same parents, so as to solve the nano-carrier of PEGylation, to enter born of the same parents' efficiency low, and enter after born of the same parents effectively to release the drug problem in intracellular.The carrier surface can modify the antibody or part of tumour-specific, further improve tumor-targeting, strengthen therapeutic effect.

Description

The sensitive targeting phosphatide polyhistidyl nanoparticles of pH for containing antineoplastic

Technical field

The present invention relates to a kind of sensitive targeting phosphatide polyhistidyl nanoparticles of pH for being used to contain cancer therapy drug, it is intended to increases Anticancer therapeutic and reduction toxic side effect, the treatment for tumour.

Background technology

Malignant tumour is one of the main reason for causing human death.Chemotherapy is that current human treatment's malignant tumour is conventional Method.However, most chemotherapeutics while killing tumor cell, can also produce without selectivity to normal cell Raw lethal effect, causes serious toxic side effect, the induced cardiotoxicity of such as adriamycin.In recent years, nanometer formulation (lipid Body, micella, nanoparticle etc.) it is widely used in terms of oncotherapy.Research shows that particle diameter is received below 200nm's Rice corpuscles, high-permeability and retention effect (the Enhanced permeability and retention of solid tumor can be passed through Effect, EPR) drug targeting is delivered to tumor locus, whole body toxic side effect is reduced, improves therapeutic index.In addition, PEGylation Nanometer formulation reticuloendothelial system (Reticuloendothelial can also be effectively shielded from blood circulation System, RES) capture, extend time for circulating in vivo, so as to improve bioavilability.For example, using phospholipid material structure The liposome (Liposomes) built is the most commonly used nanometer formulation of current clinical practice, has that internal stability is good, biofacies The advantages that capacitive is good, drugloading rate is high, tumor-targeting, and surface modification polyethylene glycol (PEG) has long circulating characteristic afterwards.Ah Mycin liposome (DOXIL) is the liposome medicament of first FDA approval listing, clinically applies for more than 20 years.

But the existing nanometer formulation clinically applied, such as Evacet (DOXIL), although can effectively carry High tolerance dose in vivo and reduce general toxicity, but Long-term clinical test result indicates that, its oncotherapy effect is not aobvious Write better than free adriamycin.Research shows, the nano-carrier of PEGylation is slow in tumor locus drug release, and can not effective quilt Tumour cell is absorbed, and the nano-carrier after the intake of part can not also escape effectively from lysosome to be released medicine into endochylema, Cause Intracellular drug to reach effective treatment concentration, have a strong impact on its therapeutic effect.

The intelligent response type nanometer built using the difference of tumor microenvironment and normal physiological context pH, temperature, enzyme etc. is carried Body is that solve the important method that nano-carrier enters born of the same parents and the problem that releases the drug, of great interest.For example, the cell of normal structure Outer liquid pH is 7.4, and the pH outside tumour cell will be less than normal structure, and its acid range is up to 7.0-6.5, and the Asia of tumor tissues The pH of cellular level is lower, if late period endosome and lysosome are up to 6.5-4.5.Utilize the subacidity of tumor tissues and lysosome Nano-carrier prepared by environment, intake and release of the medicine in tumor locus can be effectively increased, help to realize the lyase of medicine Body is escaped, and improves therapeutic effect.Wherein, polyhistidyl (Poly-Histidine, PHIS) is a kind of poly- with pH sensitiveness Compound, the characteristics of lone pair electrons on unsaturated nitrogen atom on its imidazole ring make it have Protonation-deprotonation.Its proton A large amount of protons are captured after change, produce proton sponge effect, destroy the membrane structure of the subcellular structure such as endosome and lysosome.Research Show, the micella prepared using polyhistidyl can effectively realize that the lysosome of medicine is escaped.

Active targeting nanometer formulation refers in carrier surface modified antibodies, part, cell-penetrating peptide equimolecular, by antibody with swelling Oncocyte surface specific antigen binding, part is combined with tumor cell surface specific receptor or cell-penetrating peptide and the phase of cell membrane Interaction, increase carrier is in the accumulation of tumor tissues and adhesion and intake with tumor cell membrane.Although such production is there is no at present Product list, but existing minority enters clinical test, such as the oxaliplatin liposome of targeting Surface-modified by Transferrin has been enter into II Phase is clinical.

The content of the invention

It is an object of the invention to provide a kind of pH sensitivity phosphatide polyhistidyl nanoparticles for being used to contain antineoplastic (Lipid poly-L-histidine hybrid nanoparticles, LPNs), can be used for the treatment of various cancers. LPNs is based on a kind of novel nano delivery system for designing to obtain on the basis of liposome and nanoparticle.LPNs is a kind of with poly- It is hydrophobic cores that histidine, which contains insoluble anti-tumor medicament, and individual layer phosphatide is the core-shell type nano grain of shell, surface modification There are PEG and tumor specific antibody or part.PH sensitivities LPNs has the liposome bio-compatible of similar PEGylation good, stably The characteristics such as high, the internal long circulating of property.Its pH sensitiveness makes LPNs that current potential upset occur in tumor microenvironment, is changed into connecing from negative electricity Weakly acidic pH, promote carrier to absorb into born of the same parents, lysosome escape, effectively rapid delivery of pharmaceuticals, killing are mediated in lysosome after entering born of the same parents Tumour cell, so as to solve the nano-carrier of PEGylation, to enter born of the same parents' efficiency low, enters after born of the same parents and can not be carried effectively intracellular releases the drug the problems such as The therapeutic effect of high medicine.Also, LPNs is easy to modify in its surface, can be obtained by the antibody or part for modifying tumour-specific The pH sensitivity LPNs of active targeting, tumor-targeting is improved, further enhances therapeutic effect.

The sensitive targeting phosphatide polyhistidyl nanoparticles of pH provided by the present invention for containing cancer therapy drug, the matter of its raw material Measure percentage composition：

Polyhistidyl 50~80%

Phosphatide (including phosphatide-PEG) 20%~50%

Wherein, phosphatide-PEG (PEG-DSPE) accounts for the 1%~100% of phosphatide gross mass.

Described polyhistidyl accounts for the 30% of raw material gross mass；Phosphatide-PEG accounts for the 75% of phosphatide gross mass.

The particle diameter of the described sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug is 50-200nm, It is preferred that 100nm.

Described phosphatide is selected from：Egg yolk lecithin (EPC), soybean lecithin (SPC), DLPC (DLPC), two Myristoyl lecithin (DMPC), DPPC (DPPC), distearoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), MPPC (MPPC), PMPC (PMPC), 1- palms Acyl -2- stearoyls lecithin (PSPC), SPPC (SPPC), hydrogenated soybean lecithin (HSPC), dioleoyl Base lecithin (DOPC), PE (DLPG), two palm phosphatidyl glycerols (DPPG), distearoylphosphatidyl are sweet Oily (DSPG), DOPG (DOPG), two myristoyl phosphatidic acids (DMPA), DPPA (DPPA), DMPEA (DMPE), DPPE (DPPE), two myristoyl phosphatidyl silk ammonia Sour (DMPS), the serine of two palmityl phosphatidyl two (DPPS), cephalin acyl serine (PS), cranial nerve sphingomyelins (BSP), two Palmityl sphingomyelin (DPSP), distearyl sphingomyelin (DSSP), DSPE (DSPE) it It is one or more.It is preferred that egg yolk lecithin (EPC).

The preparation method of pH sensitivity phosphatide polyhistidyl nanoparticles provided by the present invention for containing antineoplastic can It is prepared by a step precipitation method, emulsification volatility process, dialysis, specifically includes step in detail below：

1) LPNs is prepared using a step precipitation method, weighing polyhistidyl by metering precision, to be dissolved in anhydrous dimethyl sulphoxide molten In liquid, 2h is stirred, as organic phase；Phosphatide and phosphatide-PEG are weighed, is placed in the 2mM borax solns of 4% ethanol, in 65 DEG C of water Stirred in bath, as aqueous phase.Then by organic phase, (800-1200rpm, preferably 1000rpm) instills aqueous phase under high velocity agitation, Remove water-bath and be slowly dropped to room temperature and persistently stir 2h；Finally it is fitted into bag filter (3500kDa) to be placed in 2mM borax dialyzates, 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature.Resulting solution is prepared LPNs solution in bag filter；Or

2) LPNs is prepared using emulsification volatility process, weighs polyhistidyl and phosphatide and phosphatide-PEG by metering precision, dissolve In chloroform, 2h is stirred.Then chloroformic solution is added drop-wise in the 2mM borax solns that quickly stirred, obtained white Color emulsion.Emulsion is then stirred to volatilization 2h in fume hood, obtains clear solution.Solution is finally loaded into bag filter (3500kDa) is placed in 2mM borax dialyzates, and 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature, and gained is molten in bag filter Liquid is prepared LPNs solution；Or

3) LPNs is prepared using dialysis, weigh polyhistidyl and phosphatide by metering precision and phosphatide-PEG be dissolved in it is anhydrous In dimethyl sulfoxide (DMSO), 2h is stirred.Anhydrous dimethyl sulphoxide solution loading bag filter (3500kDa) will be obtained and be placed in 2mM boron 1h, 2h, 4h, 8h, 12h and 24h change extracellular fluid dialysis at room temperature in sand dialyzate.It is prepared LPNs in last bag filter Solution.

The molecular weight of described polyhistidyl is 500~10000, preferably 3000.

The molecular weight of described polyethylene glycol is 500~20000, preferably 2000.

The sensitive targeting phosphatide polyhistidyl nanoparticles of pH provided by the present invention for containing cancer therapy drug, its surface modification The part or antibody of tumour-specific are selected from：NGR(CNGRCVSGCAGRC)、RGD(CDCRGDCFC)、iRGD(CRGDKGPDC)、 INGR (CRNGRGPDC), FA (folic acid), transferrins, HAIYPRH, YSAYPDSVPMMS (YSA), ATWLPPR, F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK)、Esbp(DITWDQLWDLMK)、IELLQAR、F56 (WHSDMEWWYLLG)、K237(HTMYYHHYQHHL)、A7R(ATWLPPR)、

CSCKNTDSRCKARQLELNERTCRC、CREKA、TCP-1(CTPSPFSHC)、TAASGVRSMH, LTLRWVGLMS、KAA(CKAAKNK)、RSR(CRSRKG)、RGR(CRGRRST)、LyP-1(CGNKRTRGC)、LyP-2(LyP- 2)、GE11(YHWYGYTPQNVI)、F56(WHSDMEWWYLLG)、P1(CSDSWHYWC)、CRTIGPSVC、SPRPRHTLRLSL、 LTVSPWY、LTVSPWY、YCAREPPTRTFAYWG、WHPWSYLWTQQA(RP-1)、RLVSYNGIIFFLK、VLWLKNR、 ANTPCGPYTHDCPVKR、PQNSKIPGPTFLDPH、CTVALPGGYVRVC、CSNRDARRC、CSSRTMHHC、 LLADTTHHRPWT、c-dGHCitGPQ-c、VRPMPLQ、YEQDPWGVKWWY、LSLERFLRCWSDAPA、FYPSYHSTPQRP、 VTLTYEFAAGPRD、Anti-HER2 Fab′、Anti-ErbB2 scFv、αCD19 orαCD20mAb、Anti-rat CC531mAb、rhuMAb HER2-Fab’or scFv C6.5、F(ab’)₂of GAH、C225-Fab’、anti-EGFR scFv C10、Fab’of C225 mAb(cetuximab)、Fab’of matuzumab or C225、Anti-HER2 Fab’or scFv F5、mAb 2C5、scFv(HD37-CCH)、HD37Fab′、CD19 mAb HD37、Trastuzumab or rituximab、 OX26 mAb、Anti-CD19 mAb、Anti-CD20mAb、Antinucleosome mAb 2C5、Anti-CD19 or anti- CD20 plus anti-CD37 mAb etc..

It is preferred that NGR (CNGRCVSGCAGRC), RGD (CDCRGDCFC), iRGD (CRGDKGPDC), iNGR (CRNGRGPDC), FA (folic acid), transferrins, particularly preferred iNGR.

Described iNGR (CRNGRGPDC), iNRG include two sequence motifs：Motif NRG can be with target tumor Tissue Blood Pipe and tumor cell surface CD13；Recessive CendR modification can increase infiltration of the nano particle to tumor tissues, and they are successful Anticancer drugs, doxorubicin is improved to the therapeutic effects of high metastatic mouse source breast cancer 4T1 mouse, solves medicine from blood vessel to swollen The problem of tumor tissue infiltration is insufficient.The micella modified with iNGR, is not only significantly increased antitumous effect after containing taxol, also subtracted Few medicine whole body dosage, reduces adverse drug reaction.Therefore, research selection iNGR modifications LPNs, it is intended to realize nanometer formulation pair The active targeting administration of tumor tissues, improve the intake of infiltrations and tumour cell of the LPNs to tumor tissues.

The described sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug, surface modification tomour specific Property part or antibody ratio be phosphatide-PEG gross masses 1-100%.

The sensitive targeting phosphatide polyhistidyl nanoparticles of pH provided by the present invention for containing cancer therapy drug, what it was contained dredges The cancer therapy drug of water is selected from：Adriamycin, Epi-ADM, cis-platinum, daunorubicin, taxol, docetaxel, camptothecine or hydroxyl happiness Set alkali etc..Adriamycin is clinically more conventional broad-spectrum anti-cancer drug, and its action target spot is located in nucleus.Contain anticancer The content of medicine be preparation gross mass 1-20%, preferably 8%~10%.

The sensitive targeting phosphatide polyhistidyl nanoparticles of pH provided by the present invention for containing cancer therapy drug, its preparation method Including step in detail below：

1) LPNs for containing medicine is prepared using a step precipitation method.Polyhistidyl is weighed by metering precision and contains cancer therapy drug It is dissolved in anhydrous dimethyl sulphoxide solution, 2h is stirred, as organic phase.Phosphatide and phosphatide-PEG are weighed, is placed in 4% ethanol In 2mM borax solns, in 65 DEG C of stirred in water bath, as aqueous phase.Then by organic phase under high velocity agitation (800-1200rpm, It is preferred that 1000rpm) aqueous phase is instilled, remove water-bath and be slowly dropped to room temperature and persistently stir 2h.Finally load bag filter (3500kDa) It is placed in 2mM borax dialyzates, 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature.Resulting solution is institute in bag filter The LPNs solution for containing medicine prepared.

2) prepared using emulsification volatility process and carry medicine LPNs.By metering precision weigh polyhistidyl, phosphatide and phosphatide-PEG and Cancer therapy drug is dissolved in chloroform, is stirred 2h.It is molten that chloroformic solution is then added drop-wise to the 2mM boraxs that quickly stirred In liquid, white emulsion is obtained.Emulsion is then stirred to volatilization 2h in fume hood, obtains clear solution.Finally solution is loaded Bag filter (3500kDa) is placed in 2mM borax dialyzates, and 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature.In bag filter Resulting solution is the prepared LPNs solution for containing cancer therapy drug.

3) prepared using dialysis and carry medicine LPNs.Polyhistidyl, phosphatide, phosphatide-PEG are weighed by metering precision and are contained anti- Cancer drug is dissolved in anhydrous dimethyl sulphoxide, is stirred 2h.Anhydrous dimethyl sulphoxide solution will be obtained and load bag filter (3500kDa) is placed in 2mM borax dialyzates 1h, 2h, 4h, 8h, 12h and 24h at room temperature and changes extracellular fluid dialysis.Last bag filter Interior is the prepared LPNs solution for containing cancer therapy drug.

By usual way LPNs lyophilized formulations are made in the LPNs solution for containing cancer therapy drug by the present invention.

The pH of part or antibody provided by the present invention for its surface modification tumour-specific for containing cancer therapy drug is quick The step of sense targeting LPNs preparation method includes：Part or antibody pass through phosphatide (such as DSPE-PEG) phase for being modified with PEG Even, lead compound is synthesized, then adds in the above-mentioned precipitation method, emulsification volatility process or dialysis preparation prescription and makes simultaneously with phosphatide .Lead compound synthesizes：Take certain mol proportion (1.5:1) phosphatide-PEG-NHS (phosphatide Active Ester of Polyethyen Glycol) and targeting Antibody or part, are solvent with anhydrous dimethyl sulphoxide, and triethylamine regulation pH to 8 is reacted, tracked using thin-layer chromatography TLC anti- Should；Reaction carries out dialysis purification after terminating, and the product of gained is placed in bag filter (MWCO 3500kDa), dialysed at room temperature； Dialysis is freeze-dried to obtain fluffy white powder after terminating；Product with methanol dissolving carry out MALDI-TOF mass spectral analyses come Lead compound structure is confirmed, remaining is in -20 DEG C of freezen protectives.

The sensitive targeting phosphatide polyhistidyl nanoparticles of pH provided by the present invention for containing cancer therapy drug, its feature are：

1) the advantages that having the biocompatibility of liposome, internal stability, long circulating concurrently, while can effectively realize that tumour is thin The intake of born of the same parents and lysosome escape；

2) similar pH sensitivity micellas are compared, it has more preferable stability, and a step precipitation method can be used to prepare, letter It is easily feasible, it is suitable for industrialized large-scaled production；

3) surface is convenient for various modifications, can target different tumours by modifying different part or antibody, have wide General adaptability.

4) pair high transfevent breast cancer insensitive with chemotherapy, the delivery system have notable antitumor action effect inside and outside Fruit, shows as decreased tumor growth compared with control group, and survival rate improves.The proliferation activity drop of Showed by immune group result tumour Low, angiogenesis is reduced.Meanwhile tolerance is significantly improved, the toxic side effect of medicine is reduced, such as the cardiac toxic of adriamycin.

Brief description of the drawings

Fig. 1 LPNs of the present invention grain-size graph.

Fig. 2 LPNs of the present invention transmission electron microscope picture.

Fig. 3 DSPE-PEG-iNGR mass spectrogram.

Fig. 4 LPNs of the present invention DOX cumulative releases figures under different pH.

Fig. 5 LPNs of the present invention change of size figures under different pH.

Fig. 6 LPNs potential change figures under different pH.

Fig. 7 flow cytometers determine cellular uptake.It is all the free of 10ug/mL to be incubated concentration simultaneously in pH 6.8 and 7.4 Adriamycin (DOX), free adriamycin+free iNGR (DOX+iNGR), carry adriamycin LNPs (LPNs/DOX) and iNGR modifications After the culture 2h for carrying adriamycin LNPs (iNGR-LPNs/DOX), the streaming result of the intracellular doxorubicin contents of 4T1 is quantitatively schemed.*P< 0.05；**P<0.01.

Fig. 8 cellulotoxic experiments.The free adriamycins (DOX) of various concentrations, the LNPs (LPNs/DOX) for carrying adriamycin and The survival rate figure of 4T1 cells under the LNPs (iNGR-LPNs/DOX) of the load adriamycin of iNGR modifications.*P<0.05；**P<0.01.

Distribution experiments in Fig. 9 bodies.Give the LPNs (iNGR-LPNs) that DiR dosage is 0.4ug/100 μ L/ iNGR modifications With LPNs lotus knurl female BAl BIc/c nude mice different time sections living imaging figure.

Figure 10 Tumor growth inhibitions are tested.200uL PBS, the DOX (5mg/kg) that dissociates are given respectively, carry adriamycin LNPs (iNGR-LPNs/DOX, 10mg/kg) lotus knurl of the load adriamycin of LNPs (LPNs/DOX, 10mg/kg) and iNGR modifications is female Property BALB/c mouse tumor Volume Changes figure.

Survival rate in Figure 11 bodies.200 μ L PBS, free DOX (5mg/kg), the LPNs for carrying adriamycin is given respectively LNPs (iNGR-LNPs/DOX, 10mg/kg) each group lotus knurls female BAl BIc/c of the load adriamycin of (10mg/kg) and iNGR modification Mouse survival rate figure.

The LNPs that Figure 12 gives 200 μ L PBS respectively, free DOX (5mg/kg), LPNs (10mg/kg) and iNGR are modified (iNGR-LPNS/DOX, 10mg/kg) each group lotus knurl female BAl BIc/c mouse weight variation diagrams.

The LNPs that Figure 13 gives 200 μ L PBS respectively, free DOX (5mg/kg), LPNs (10mg/kg) and iNGR are modified (iNGR-LPNS/DOX, 10mg/kg) lotus knurl female BAl BIc/c mouse CD31, Ki67 and H＆E colored graphs.

Specific embodiment

The present invention is further illustrated and explains by the following examples, but not as the limitation that the present invention is carried out.Embodiment In unreceipted actual conditions experimental method, generally according to the condition described in normal condition and handbook, or according to manufactory Condition proposed by business；Common apparatus used, material, reagent etc., unless otherwise specified, are commercially obtained.

The LPNs of the blank of embodiment 1 preparation

LPNs is prepared using a step precipitation method.Precision weighs a certain amount of polyhistidyl (Qiang Yao bio tech ltd Synthesis, molecular weight 3000,4.1mg) it is dissolved in anhydrous dimethyl sulphoxide solution, 2h is stirred, as organic phase.Weigh certain ratio Example egg yolk lecithin (EPC, pharmaceutical grade, Shanghai Advanced viecle Technology Co., Ltd.) and phosphatide-PEG (DSPE-PEG, >= 98%, Shanghai Advanced viecle Technology Co., Ltd.) (mol ratio 1:3, respectively 2.2g and 3.3g, PEG molecular weight are 2000) in the 2mM borax solns of 4% ethanol, in 65 DEG C of stirred in water bath, as aqueous phase.Then by organic phase in high-speed stirring Mix down (800-1200rpm, preferably 1000rpm) and instill aqueous phase, remove water-bath and be slowly dropped to room temperature and persistently stir 2h.Finally fill Enter bag filter (3500kDa) to be placed in 2mM borax dialyzates, 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature.Bag filter Interior resulting solution is prepared LPNs solution.Deposited in 4 DEG C of refrigerators standby, its grain is determined using Malvern particle instrument Footpath, current potential.Blank LPNs 100nm obtained above or so.As a result Fig. 1 is seen.

LPNs is prepared using emulsification volatility process.Precision weighs a certain amount of polyhistidyl (molecular weight 3000,4.1mg) and one (phospholipid material mol ratio is 1 to the EPC and DSPE-PEG of certainty ratio:3, respectively 2.2g and 3.3g, PEG molecular weight are 2000) molten Solution is stirred 2h in chloroform.Then chloroformic solution is added drop-wise in the 2mM borax solns that quickly stirred, obtained White emulsion.Emulsion is then stirred to volatilization 2h in fume hood, obtains clear solution.Solution is finally loaded into bag filter (3500kDa) is placed in 2mM borax dialyzates, and 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature.Gained is molten in bag filter Liquid is prepared LPNs solution.Deposited in 4 DEG C of refrigerators standby.

LPNs is prepared using dialysis.Precision weighs a certain amount of polyhistidyl (molecular weight 3000,4.1mg) and certain ratio (phospholipid material mol ratio is 1 to the EPC and DSPE-PEG of example:3, respectively 2.2g and 3.3g, PEG molecular weight are 2000) to be dissolved in In anhydrous dimethyl sulphoxide, 2h is stirred.Anhydrous dimethyl sulphoxide solution loading bag filter (3500kDa) will be obtained to be placed in 1h, 2h, 4h, 8h, 12h and 24h change extracellular fluid dialysis at room temperature in 2mM borax dialyzates.Resulting solution is in last bag filter For prepared LPNs solution.Deposited in 4 DEG C of refrigerators standby.

Embodiment 2 contains the LPNs of adriamycin (DOX) preparation

LPNs is prepared using a step precipitation method.Precision weighs DOX (1mg) and polyhistidyl (molecular weight 3000,4.1mg) point It is not dissolved in anhydrous dimethyl sulphoxide solution, DOX adds 1:The triethylamine alkalization 2h of 3 mol ratios.Both are mixed again 2h is stirred, as organic phase.Weigh a certain proportion of EPC and DSPE-PEG (mol ratio 1:3, respectively 2.2g and 3.3g, PEG molecular weight is 2000) in the 2mM borax soln of 4% ethanol, in 65 DEG C of stirred in water bath, as aqueous phase.Then will be organic Mutually (800-1200rpm, preferably 1000rpm) instills aqueous phase under high velocity agitation, removes water-bath and is slowly dropped to room temperature and persistently stirs Mix 2h.Finally it is fitted into bag filter (3500kDa) to be placed in 2mM borax dialyzates, 1h, 2h, 4h, 8h, 12h change dialysis at room temperature Outer liquid.Resulting solution is prepared LPNs solution in bag filter.Deposited in 4 DEG C of refrigerators standby：Determine its particle diameter, current potential And pass through efficient liquid phase (HPLC) computational envelope rate (Encapsulation efficiency, EE%) and drugloading rate (Loading Efficiency, LE%).Load DOX obtained above LNPs 100nm or so, envelop rate is more than 80%, and drugloading rate is 9% More than.The transmission electron microscope picture of the LPNs/DOX is as shown in Figure 2.

Embodiment 3 contains the LPNs of Docetaxel (PTX) preparation

Precision weighs PTX (1mg) and polyhistidyl (molecular weight 3000,4.1mg) is dissolved in DMSO solution and stirs 2h, makees For organic phase.Weigh a certain proportion of EPC and DSPE-PEG (mol ratio 1:3, respectively 2.2g and 3.3g, PEG molecular weight are 2000) in the 2mM borax solns of 4% ethanol, in 65 DEG C of stirred in water bath, as aqueous phase.Then by organic phase in high-speed stirring Mix down (800-1200rpm, preferably 1000rpm) and instill aqueous phase, remove water-bath and be slowly dropped to room temperature and persistently stir 2h.Finally fill Enter bag filter (3500kDa) to be placed in 2mM borax dialyzates, 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature.Bag filter Interior resulting solution is prepared LPNs solution.Deposited in 4 DEG C of refrigerators standby：Determine its particle diameter, current potential and by efficient Liquid phase (HPLC) computational envelope rate (Encapsulation efficiency, EE%) and drugloading rate (Loading Efficiency, LE%).Load PTX obtained above LPNs 100nm or so, more than 80%, drugloading rate exists envelop rate More than 5%.

Embodiment 4 contains the DOX peptide modified iNGR-LPNs/DOX of iNGR preparation

Lead compound DSPE-PEG-iNGR is synthesized first.Take certain mol proportion (1.5:1) phosphatide-PEG-NHS (phosphorus Fat Active Ester of Polyethyen Glycol, DSPE-PEG2000-NHS, Shanghai Advanced viecle Technology Co., Ltd.) and targeting peptides iNGR is (by force The synthesis of credit bio tech ltd), anhydrous dimethyl sulphoxide is solvent, triethylamine regulation pH to 8 or so, is reacted, use is thin Layer chromatography TLC tracking reactions.Reaction carries out dialysis purification after terminating, and the product of gained is placed in into bag filter (MWCO3500kDa) In, dialyse at room temperature.Dialysis is freeze-dried to obtain fluffy white powder after terminating.Product carries out MALDI- with methanol dissolving Lead compound structure is confirmed in TOF mass spectral analyses, and remaining is in -20 DEG C of freezen protectives.Mass spectrometry results such as Fig. 3.Product peak It is identical with DSPE-PEG-iNGR theoretical moleculars for 3961, it was demonstrated that Success in Experiment synthesizes DSPE-PEG-iNGR.

Precision weighs DOX (1mg) and polyhistidyl (molecular weight 3000,4.1mg) is dissolved separately in anhydrous dimethyl sulphoxide In solution, DOX adds 1:The triethylamine alkalization 2h of 3 mol ratios.Both are subjected to mixing 2h again, as organic phase.Weigh (phospholipid material mol ratio is 1 for a certain proportion of lecithin and DSPE-PEG-iNGR:3, respectively 2.2g and 3.3g, PEG molecule Measure as 2000) in the 2mM borax solns of 4% ethanol, in 65 DEG C of stirred in water bath, as aqueous phase.Then by organic phase in height (800-1200rpm, preferably 1000rpm) instills aqueous phase under speed stirring, persistently stirs 2h and is slowly dropped to room temperature.Finally load saturating Analysis bag is placed in 2mM borax dialyzates, and 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature.Resulting solution is in bag filter For prepared iNGR/LPNs solution.Deposited in 4 DEG C of refrigerators standby：Determine its particle diameter, current potential and pass through efficient liquid phase (HPLC) computational envelope rate (Encapsulation efficiency, EE) and drugloading rate (Loading efficiency, LE). Blank LPNs obtained above 100nm or so, envelop rate is more than 80%, and drugloading rate is more than 9%.

The step precipitation method of embodiment 5 one prepare the LPNs for containing fluorescence probe DiR

DiR is fat-soluble fluorescence probe, and progress can be acted on the tumor-targetings of LPNs in vivo visually by containing DiR Change research.Precision weighs a certain amount of DiR and polyhistidyl and is dissolved in DMSO solution.Both are subjected to mixing 2h again, As organic phase, lucifuge is operated.Weighing a certain proportion of lecithin and phosphatide-PEG, (phospholipid material mol ratio is 1:3, point Not Wei 2.2g and 3.3g, PEG molecular weight for 2000) in the 2mM borax solns of 4% ethanol, in 65 DEG C of stirred in water bath, as Aqueous phase.Then by organic phase, (800-1200rpm, preferably 1000rpm) instills aqueous phase under high velocity agitation, persistently stirs 2h and delays Slowly it is down to room temperature.Finally it is fitted into bag filter to be placed in 2mM borax dialyzates, it is outer to change dialysis by 1h, 2h, 4h, 8h, 12h at room temperature Liquid.What resulting solution was as prepared in bag filter contains LPNs solution.The iNGR-LPNs for containing DiR can be prepared with method.

Using sepectrophotofluorometer method measure DiR content in experiment.Precision weighing DiR solids, are dissolved with methanol, matched somebody with somebody Series of standards solution processed, 10,20,40,80,100,200,300,400,500ng/mL according to condition determination (λ ex=748nm, Slit width 5nm；λ em=780nm, slit width 5nm) its fluorescence intensity (Fluorescent intensity, F) is determined, keep away Light operates, and the above operates in triplicate, and each concentration configures three standard liquids.With fluorescence intensity A to DiR concentration Cs (ng/mL) Linear regression is carried out, draws standard curve.The fluorescent value of different preparations is determined, is first handled with the methanol rupture of membranes of two volumes (super Sound 5min) it is measured again.

The preparation of embodiment 6LPNs lyophilized formulations

Take and carry medicine LPNs preparations, add trehalose (or mannitol) 2-10%.In 4 DEG C of refrigerator pre-freeze 30min, -20 DEG C of refrigerators Pre-freeze 2h, then in the pre- 12h of -80 DEG C of refrigerators.Seal pipe switch is opened, moves freezing room, the dividing plate that sample is placed in freezing room On.Close seal pipe switch.Open freeze drier freeze switch, wait 20~30min, until freezing room temperature less than- 40℃.Vacuum pump switch is opened, 10~15min is waited, until system pressure is less than 100millitorr.After drying 24h, open Seal pipe switch closes freeze switch, takes out sample to release vacuum state.Redissolve, and test its particle diameter.The lyophilized front and rear transformation of the way Significant change does not occur for agent particle diameter, and medicine does not occur substantially to leak.

Embodiment 7 contains releases of the DOX LPNs under condition of different pH

Insoluble drug releases of the LPNs for containing DOX under different pH is investigated using dynamic dialysis method, carrying medicine solution and After dissolution medium (pH is respectively 7.4,6.5,5.5 PBS) preheats 1h in 37 DEG C of insulating boxs, take appropriate LPNs molten Liquid is mixed with the dissolution medium of equivalent, is placed into bag filter (MWCO=1000kDa), both ends are placed in 30mL phases after tightening In the pH answered PBS dissolution mediums, in 37 DEG C, 180rpm constant temperature oscillations.Presetting time point 0.5h, 1h, 2h, 4h, 8h, 12h, 24h take 1mL extracellular fluid dialysis respectively, while supplement corresponding pH 1mL PBS dissolution mediums, and n=3 operation repetitives carry out real Test.Experiment carries out quantitative analysis using burst size of efficient liquid phase (HPLC) the detection medicine under different pH environment, first to flow It is dynamic mutually to prepare series of standards solution：40th, 30,20,18,16,14,12,10,8,6,4 μ g/mL, and carry out HPLC measure.Line Property is returned after obtaining normal equation, and seeks the accumulative release rate for calculating each time point medicine.

HPLC chromatogram condition：Chromatographic column：C18 reverse-phase chromatographic columns (Agilent ZORBAX), SB-C18,5 μm of particle diameter, (4.6mm × 250mm, L × ID)；Mobile phase：Aqueous phase (SDS 1.44g, H₃PO₄0.68mL, 500mL ultra-pure water)：Organic phase (second Nitrile：Methanol=10:1)=65:35；Flow velocity：1.0mL/min；Column temperature：25℃；Detection wavelength：233nm；Sample size：20μL.

Experimental result：DOX LPNs drug release profiles are contained as shown in figure 4, the accumulative release rate of medicine is reachable in pH5.5 80%, it is 50% or so in pH6.5, and medicine adds up release rate only 20% or so during pH7.4.

Particle diameters and potential change of the embodiment 8LPNs under condition of different pH

The blank LPNs solution 3mL that Example 1 prepares, pH about 0.5 is lowered with 0.01M HCl solutions before measure every time Individual pH value unit, its particle diameter and current potential are determined using Malvern particle instrument, and carry out recording drawing.

Experimental result：Change of size sees Fig. 5, it is seen that with pH reduction, LPNs particle diameters become larger, in pH 7.4 100nm or so, in pH 4, nearby particle diameter becomes greatly more than 200nm, is also become larger with pH reduction polydispersity coefficient.Current potential Change is as shown in fig. 6, result is visible as pH reduction, LPNs current potential are nearby overturn as just 6.5 by negative.

Embodiment 9 contains cellular uptakes of the DOX LPNs under condition of different pH

Because the present invention is using the pH sensitiveness of the polyhistidyl in LPNs, iNGR tumor-targeting, increase cell pair LNPs intake, improve the concentration of DOX in the cell.Therefore, the intake of its cell in vitro is investigated.Use flow cytometer Investigation is under the conditions of pH is 6.8 and 7.4, mixed solution (DOX+iNGR), the load LPNs/ of the DOX that dissociates, free DOX with iNGR peptides DOX and iNGR-LPNs/DOX cellular uptake.

Mouse source breast cancer cell (4T1) is chosen in experiment, by the cell suspension inoculation in 12 orifice plates, 37 DEG C, cultivates 24h Afterwards, the uniform adherent growth of cell to cell density reaches 80%.PH be 6.8 and 7.4 nutrient solutions in be separately added into free DOX, The load adriamycin LPNs (iNGR-LPNs/DOX) for carrying LPNs (LPNs/DOX) and the iNGR modification of adriamycin is put in 37 DEG C of incubators After being incubated 2h, intracellular DOX fluorescence intensities are determined with flow cytometer.

Experimental result：Flow cytometric analysis data is shown in Fig. 7.Fruit shows that LPNs and iNGR-LPNs groups are absorbed in pH6.8 When being all higher than pH7.4, it was demonstrated that LPNs intakes have pH sensitiveness, and strengthen under tumour solutions of weak acidity；In pH6.8 and 7.4, For iNGR-LPNs/DOX compared with LPNs, intracellular fluorescence intensity is significantly stronger, it was demonstrated that being effectively increased for iNGR modifications is thin Intake of the born of the same parents to LPNs.

Embodiment 10 contains DOX LPNs to 4T1 cell growth inhibitions

Experiment is studied using mtt assay.Take the logarithm the 4T1 cells of growth, with 5 × 10³It is individual/three piece 96 is added per hole Orifice plate, it is put into 37 DEG C of cell culture incubators.After cultivating 36h, original nutrient solution is discarded.Every piece of 96 orifice plates add：Free DOX, LPNs/DOX and iNGR-LPNs/DOX, a series of every group of decoction that various concentrations are prepared with pH 6.8 nutrient solution dilution, every group If 9 concentration gradients:0、0.01、0.1、0.5、1、2、5、10μg/mL.Concentration gradient sets 6 multiple holes, per hole culture medium cumulative volume For 100 μ L, and 12 negative control holes (cell nature growth group) and 2 zeroing holes (200 μ L nutrient solutions) are set, cell is 37 DEG C, 5%CO₂Under the conditions of continue cultivate 24h.

5mg/mLMTT solution 20 μ L are added per hole, 4h is stood at 37 DEG C, MTT solution is abandoned in suction, and DMSO200 μ L are added per hole, 37 DEG C of low-speed oscillation 10min on air table are put, trap of each hole under 490nm wavelength is determined with ELIASA.Record result, Calculate cell proliferation inhibition rate under different pharmaceutical concentration.

Experimental result：Cell growth inhibition assay result is shown in Fig. 8 and table 1.As a result visible iNGR-LPNs/DOX IC₅₀It is small In LPNs/DOX, close to free DOX, it is seen that iNGR modification promotes intake of the cell to LPNs.

Table 1

Embodiment 11 contains the targeting distribution of fluorescence probe DiR LPNs and iNGR/LPNs in nude mouse

Due to present invention utilizes the targeting that iNGR can strengthen nanoparticle, therefore, carrying out targeting in animal body Research has great significance.Living imaging experiment can be loaded with the liposome of fluorescence probe in tumor bearing nude mice body with real-time monitored Distribution.High metastatic breast cancer cell 4T1 is incubated in DMEM complete culture solutions, blake bottle half opening is placed in into 37 DEG C, relatively Humidity is 95%, CO₂In the cell culture incubator that content is 5% after cell confluency to 90%, digested, terminated with 0.25% pancreatin It is transferred in centrifuge tube and centrifuges after digestion, removes supernatant, cell is suspended in the nutrient solution without serum again, is made Single cell suspension.Take 100 μ L cell suspensions (1 × 10⁶It is individual) oxter on the right side of female BAl BIc/c nude mices is inoculated in, (5-7 days) wait to swell Knurl volume reaches about 100mm³It can be tested.It is female BAl BIc/c the nude mices for being inoculated with 4T1 below, tail vein injection contains Fluorescent dye DiR LPNs and iNGR-LPNs experimental result (see Fig. 9).As a result show, iNGR-LPNs group tumor-targetings It is better than LPNs groups.

Embodiment 12 contains DOX LPNs pharmacodynamics test and preliminary toxicity is investigated

Reference preparation：DOX solution

Test preparation：LPNs/DOX and iNGR-LPNs/DOX

Experimental animal：Inoculation inoculation 4T1 female BAl BIc/c mouse, 18-22 grams of body weight, every group of number of mice is 6, and tail is quiet Arteries and veins drug administration by injection, inspection target are gross tumor volume.

Administering mode：It is administered within 10th, 13,16,20,23 day.

Result of the test：Result of the test is shown in accompanying drawing 10 and 11.

As a result show：Figure 10 results are visible, and compared with control group, LPNs/DOX and iNGR-LPNs/DOX groups can be obvious Reduce gross tumor volume, suppress the growth of tumour.Figure 11 is visible, and LPNs/DOX and iNGR-LPNs/DOX extend being averaged for mouse Survival rate.Figure 13 is visible, and SABC CD 31 and the dyeing of Ki 67 show that LPNs/DOX groups and iNGR-LPNs/DOX groups represent newly Angiogenic and the brown color of proliferative cell are considerably less than PBS groups, DOX groups.

The preliminary toxicity research of preparation, as seen from Figure 12, compared with control group, high dose LPNs/DOX and iNGR-LPNs/ The body weight of DOX groups does not have significant change, and death occurs in the free DOX groups mouse of Isodose in preliminary experiment.Illustrate that LPNs is carried High DOX tolerance.Figure 13 is visible, and heart H＆E coloration results are visible, and LPNs/DOX and iNGR-LPNs/DOX be not notable Cardiac toxic, and the muscle fibril tissue and inflammatory infiltration of unicellular necrosis, cytoplasmic vacuoles and large area is presented in DOX groups, Certain cardiac toxic is presented.

In testing above, the present invention is only the load DOX prepared in the selected part embodiment of example LPNs conducts For experimental drug, it is necessary to explanation, load other medicines of the invention or the LPNs of modified antibodies or part also have identical or phase Near beneficial effect.Therefore, with the LPNs containing medicine of the present invention or modified antibodies or part also by the present invention's Within protection domain.

Claims (9)

1. A kind of 1. sensitive targeting phosphatide polyhistidyl nanoparticles of pH for being used to contain cancer therapy drug, it is characterised in that the matter of its raw material Measure percentage composition：

   Polyhistidyl 50~80%

   Phosphatide (including phosphatide-PEG) 20%~50%

   Wherein, phosphatide-PEG accounts for the 1%~100% of phosphatide gross mass.
2. 2. according to the sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug described in claim 1, it is special Sign is that described polyhistidyl accounts for the 30% of raw material gross mass；Phosphatide-PEG accounts for the 75% of phosphatide gross mass；Described pH is quick The particle diameter of sense targeting phosphatide polyhistidyl nanoparticle is 50-200nm.
3. 3. according to the sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug described in claim 1, it is special Sign is that described phosphatide is selected from：Egg yolk lecithin (EPC), soybean lecithin (SPC), DLPC (DLPC), two meat Myristoyl lecithin (DMPC), DPPC (DPPC), distearoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), MPPC (MPPC), PMPC (PMPC), 1- palms Acyl -2- stearoyls lecithin (PSPC), SPPC (SPPC), hydrogenated soybean lecithin (HSPC), dioleoyl Base lecithin (DOPC), PE (DLPG), two palm phosphatidyl glycerols (DPPG), distearoylphosphatidyl are sweet Oily (DSPG), DOPG (DOPG), two myristoyl phosphatidic acids (DMPA), DPPA (DPPA), DMPEA (DMPE), DPPE (DPPE), two myristoyl phosphatidyl silk ammonia Sour (DMPS), the serine of two palmityl phosphatidyl two (DPPS), cephalin acyl serine (PS), cranial nerve sphingomyelins (BSP), two Palmityl sphingomyelin (DPSP), distearyl sphingomyelin (DSSP), DSPE (DSPE) it It is one or more；It is preferred that egg yolk lecithin (EPC).
4. 4. the preparation side of the sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug described in claim 1 Method, it is characterised in that including step in detail below：

   1) LPNs is prepared using a step precipitation method, weighing polyhistidyl by metering precision is dissolved in anhydrous dimethyl sulphoxide solution In, 2h is stirred, as organic phase；Phosphatide and phosphatide-PEG are weighed, is placed in the 2mM borax solns of 4% ethanol, in 65 DEG C of water-baths Middle stirring, as aqueous phase.Then by organic phase, (800-1200rpm, preferably 1000rpm) instills aqueous phase under high velocity agitation, removes Go water-bath and be slowly dropped to room temperature persistently to stir 2h；Finally it is fitted into bag filter (3500kDa) to be placed in 2mM borax dialyzates, room Temperature lower 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis.Resulting solution is prepared LPNs solution in bag filter；Or

   2) LPNs is prepared using emulsification volatility process, weighs polyhistidyl and phosphatide and phosphatide-PEG by metering precision, be dissolved in three In chloromethanes, 2h is stirred.Then chloroformic solution is added drop-wise in the 2mM borax solns that quickly stirred, obtains white breast Liquid.Emulsion is then stirred to volatilization 2h in fume hood, obtains clear solution.Solution is finally loaded into bag filter (3500kDa) It is placed in 2mM borax dialyzates, 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature, and resulting solution is institute in bag filter The LPNs solution of preparation；Or

   3) LPNs is prepared using dialysis, weighs polyhistidyl and phosphatide by metering precision and phosphatide-PEG is dissolved in anhydrous dimethyl In base sulfoxide, 2h is stirred.Anhydrous dimethyl sulphoxide solution will be obtained to load bag filter (3500kDa) to be placed in 2mM boraxs saturating 1h, 2h, 4h, 8h, 12h and 24h change extracellular fluid dialysis at room temperature in analysis liquid.It is prepared LPNs molten in last bag filter Liquid.
5. 5. according to the preparation method described in claim 4, it is characterised in that the molecular weight of described polyhistidyl be 500~ 10000, preferably 3000；The molecular weight of described polyethylene glycol is 500~20000, preferably 2000.
6. 6. any described sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug of claim 1-3, its Being characterised by surface modification has tumor specific antibody or a part, the part of described surface modification tumour-specific or antibody Ratio is the 1-100% of phosphatide-PEG gross masses；

   Tumor specific antibody or part are selected from：NGR(CNGRCVSGCAGRC)、RGD(CDCRGDCFC)、iRGD (CRGDKGPDC), iNGR (CRNGRGPDC), FA (folic acid), transferrins, HAIYPRH, YSAYPDSVPMMS (YSA), ATWLPPR、F3(KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK)、Esbp(DITWDQLWDLMK)、IELLQAR、F56 (WHSDMEWWYLLG)、K237(HTMYYHHYQHHL)、A7R(ATWLPPR)、CSCKNTDSRCKARQLELNERTCRC、 CREKA、TCP-1(CTPSPFSHC)、TAASGVRSMH,LTLRWVGLMS、KAA(CKAAKNK)、RSR(CRSRKG)、RGR (CRGRRST)、LyP-1(CGNKRTRGC)、LyP-2(LyP-2)、GE11(YHWYGYTPQNVI)、F56(WHSDMEWWYLLG)、 P1(CSDSWHYWC)、CRTIGPSVC、SPRPRHTLRLSL、LTVSPWY、LTVSPWY、YCAREPPTRTFAYWG、 WHPWSYLWTQQA(RP-1)、RLVSYNGIIFFLK、VLWLKNR、ANTPCGPYTHDCPVKR、PQNSKIPGPTFLDPH、 CTVALPGGYVRVC、CSNRDARRC、CSSRTMHHC、LLADTTHHRPWT、c-dGHCitGPQ-c、VRPMPLQ、 YEQDPWGVKWWY、LSLERFLRCWSDAPA、FYPSYHSTPQRP、VTLTYEFAAGPRD、Anti-HER2 Fab′、Anti- ErbB2 scFv、αCD19 orαCD20mAb、Anti-rat CC531mAb、rhuMAb HER2-Fab’or scFv C6.5、F (ab’)₂ of GAH、C225-Fab’、anti-EGFR scFv C10、Fab’of C225 mAb(cetuximab)、Fab’of matuzumab or C225、Anti-HER2 Fab’or scFv F5、mAb 2C5、scFv(HD37-CCH)、HD37 Fab′、 CD19 mAb HD37、Trastuzumab or rituximab、OX26 mAb、Anti-CD19 mAb、Anti-CD20 mAb、 Antinucleosome mAb 2C5、Anti-CD19 or anti-CD20 plus anti-CD37 mAb；

   It is preferred that NGR (CNGRCVSGCAGRC), RGD (CDCRGDCFC), iRGD (CRGDKGPDC), iNGR (CRNGRGPDC), FA (folic acid), transferrins, particularly preferred iNGR.
7. 7. according to the sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug described in claim 6, it is special Sign is that its hydrophobic cancer therapy drug contained is selected from：Adriamycin, Epi-ADM, cis-platinum, daunorubicin, taxol, more west he Match, camptothecine or HCPT；Contain cancer therapy drug content be preparation gross mass 1-20%, preferably 8%~10%.
8. 8. the preparation side of the sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug described in claim 6 Method, it is characterised in that including step in detail below：

   1) LPNs for containing medicine is prepared using a step precipitation method, polyhistidyl is weighed by metering precision and contains cancer therapy drug dissolving In anhydrous dimethyl sulphoxide solution, 2h is stirred, as organic phase, phosphatide and phosphatide-PEG is weighed, is placed in the 2mM of 4% ethanol In borax soln, in 65 DEG C of stirred in water bath, as aqueous phase, then by organic phase under high velocity agitation (800-1200rpm, it is excellent Select 1000rpm) aqueous phase is instilled, remove water-bath and be slowly dropped to room temperature and persistently stir 2h, finally load bag filter (3500kDa) and put In 2mM borax dialyzates, 1h, 2h, 4h, 8h, 12h change extracellular fluid dialysis at room temperature, and resulting solution is as made in bag filter The standby LPNs solution for containing medicine；Or

   2) prepared using emulsification volatility process and carry medicine LPNs, polyhistidyl, phosphatide and phosphatide-PEG and anticancer are weighed by metering precision Medicine is dissolved in chloroform, is stirred 2h, and chloroformic solution is then added drop-wise to the 2mM borax solns that quickly stirred In, obtain white emulsion.Emulsion is then stirred to volatilization 2h in fume hood, obtains clear solution, is finally loaded solution saturating Analysis bag (3500kDa) is placed in 2mM borax dialyzates, at room temperature 1h, 2h, 4h, 8h, 12h replacing extracellular fluid dialysis, institute in bag filter It is the prepared LPNs solution for containing cancer therapy drug to obtain solution；Or

   3) prepared using dialysis and carry medicine LPNs, weighed polyhistidyl, phosphatide, phosphatide-PEG by metering precision and contain anticarcinogen Thing is dissolved in anhydrous dimethyl sulphoxide, is stirred 2h, will be obtained anhydrous dimethyl sulphoxide solution and is loaded bag filter (3500kDa) is placed in 2mM borax dialyzates 1h, 2h, 4h, 8h, 12h and 24h at room temperature and changes extracellular fluid dialysis, last bag filter Interior is the prepared LPNs solution for containing cancer therapy drug.
9. 9. according to the sensitive targeting phosphatide polyhistidyl nanoparticles of the pH for being used to contain cancer therapy drug described in claim 7, it is special Sign is the step of its preparation method includes：

   Part or antibody synthesize lead compound, then add above-mentioned sink simultaneously with phosphatide by being connected with the phosphatide that PEG is modified It is made in shallow lake method, emulsification volatility process or dialysis preparation prescription；

   Lead compound synthesizes：Take mol ratio 1.5:1 phosphatide-PEG-NHS and targeting antibodies or part, it is sub- with anhydrous dimethyl base Sulfone is solvent, and triethylamine regulation pH to 8 is reacted, tracked and reacted using thin-layer chromatography TLC；Reaction carries out dialysis purification after terminating, The product of gained is placed in bag filter (MWCO 3500kDa), dialysed at room temperature；Dialysis is freeze-dried to obtain after terminating Fluffy white powder；Product with methanol dissolving carries out MALDI-TOF mass spectral analyses to confirm lead compound structure, remaining in- 20 DEG C of freezen protectives.