CN110314136A - A kind of preparation and its application of the tumor-targeting drug based on unsaturated fatty acid nanoparticle - Google Patents

A kind of preparation and its application of the tumor-targeting drug based on unsaturated fatty acid nanoparticle Download PDF

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CN110314136A
CN110314136A CN201910356708.3A CN201910356708A CN110314136A CN 110314136 A CN110314136 A CN 110314136A CN 201910356708 A CN201910356708 A CN 201910356708A CN 110314136 A CN110314136 A CN 110314136A
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micella
peg
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dha
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CN110314136B (en
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吴宝艳
薛永永
关燕清
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South China Normal University
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Abstract

The invention discloses the preparation and its application of a kind of tumor-targeting drug based on unsaturated fatty acid nanoparticle, the present invention carries out condensation reaction using the carboxyl of docosahexaenoic acid and the amino of amino-polyethyleneglycols and micella mPEG-DHA is prepared；Utilize the carboxyl and Anti-HER of DSPE-PEG-COOH2Amino carry out condensation reaction obtain micella DSPE-PEG-Anti-HER2；Further utilize micella mPEG-DHA, micella DSPE-PEG-Anti-HER2, Methyl Pyropheophorbide and adriamycin self assembly obtain for lipid peroxidation, light power-chemotherapy combined oncotherapy targeted nano-particle DPSPAH/MPPa/DOX.The nanoparticle DPSPAH/MPPa/DOX partial size invented is in 190nm or so, stability is good, the release of chemotherapeutics can be effectively controlled, with good biocompatibility, high-efficiency low-toxicity is with high application prospect in the fields such as nano-medicament carrier and Nano medication oncotherapy.

Description

A kind of preparation of tumor-targeting drug based on unsaturated fatty acid nanoparticle and its Using
Technical field
The present invention relates to Nano medication technical field, more particularly, to a kind of based on unsaturated fatty acid nanoparticle The preparation and its application of tumor-targeting drug.
Background technique
Breast cancer is one of the malignant tumour that women is common in worldwide, and number of the infected is located at female malignant It is the first.Its disease incidence is on the rise in recent years, is that the second largest cause of the death of lung cancer is only second in malignant tumour, and age of onset year Lightization trend seriously threatens the life and health of patient.
Breast cancer is using operative treatment as essential therapeutic arsenals at present, and with radiotherapy, chemotherapy, endocrine therapy and light power The auxiliary such as treatment.Although current treatment method is fairly perfect, most of breast cancer still suffer from easy to recur, cancer cell diffusion The problem of with transfer, it is desirable to which thoroughly radical cure breast cancer is difficult to accomplish with current treatment means.Single oncotherapy means are very Difficulty is captured, and the combination therapy of tumour has become focus concerned by people.And more and more research, which demonstrates tumour and combines, controls The validity for the treatment of.
Breast cancer includes a variety of heterogeneous types, and can be divided into different Asias according to different histology and characterization of molecules Type.HER2 type breast cancer clinically accounts for about 20%~30% ratio, but since the property of HER2 proto-oncogene multicopy makes Obtaining the type tumor patient has the characteristics that survival rate is low, grade malignancy is high, progression of the disease is rapid, easy to recur and clinical prognosis is poor. Therefore there is an urgent need to a kind of tumor combined therapeutic methods for HER2 positive breast cancer.
Nanotechnology brings new breakthrough to the combination therapy of tumour.Various nanometer systems include but is not limited to nanometre glue Beam, liposome, magnetic grain, carbon nanotubes, are widely studied as pharmaceutical carrier.It is had become using nano material as pharmaceutical carrier The forward position direction of nanosecond medical science research field.Nano-micelle receives much attention in the research of pharmaceutical carrier field, contains drug side Method has: chemical bonding processes, physics contain method etc., help to realize the loading of a variety of drugs, the sustained release of drug and the guarantor of drug Shield effect etc..Micella usually has hydrophilic shell and a hydrophobic kernel, thus have good dissolubility, low cytotoxicity with And effectively drug can be avoided to be swallowed by mononuclear macrophage, the runing time of micella in blood is extended, is conducive to mention The bioavilability of high drug.
Chemotherapy is as the main means in adjuvant therapy of breast cancer, still in occupation of very important status.But it is traditional It treats drug and lacks tumor-selective, lethal effect also is generated to a part of normal cell while killing cancer cell, is thus produced Raw toxic side effect causes the dosage of chemotherapeutics and treatment cycle to be limited by the tolerance of cancer patient, and long-term Medication leads to tumor multi-medicine drug-resistant.
Photodynamic therapy is obtained in the combination therapy of tumour with the advantage and selectivity of its unique " light is removed to disease " Prominent achievement.Photodynamic therapy has normal tissue injury compared with the treatment means such as traditional operation, chemotherapy and radiation It is small, the advantages that drug resistance, repeatable treatment and Small side effects will not be generated.There are three necessary constituents for optical dynamic therapy: Photosensitizer molecule, the illumination condition and oxygen of specific wavelength.Photosensitizer molecule generates singlet oxygen after special wavelength light irradiates Irreversible damage is generated etc. a series of active oxygen species, and then to malignant tumor cells, finally makes Apoptosis/death, to reach To the purpose for the treatment of.However traditional photosensitizer water solubility is poor, is clinically difficult direct injection, is difficult effectively to reach tumour Position, and photosensitizer stability is poor, is enriched in tumor locus difficult prospect, reduces the curative effect of optical dynamic therapy, hinder The application of photodynamic therapy.Photodynamic therapy is mainly a kind of local therapeutic approaches, to the fragmentation effect of tumour very big It is decided by the laser dosage of diseased region and the amount of photosensitizer in degree.
Adriamycin has clinically obtained good therapeutic effect to the Chemotherapy of HER2 positive breast cancer, but Ah mould The problems such as element is there are poor specificity, utilization ratio of drug is low, body-internal-circulation half-life short, and can be to normal histocyte Big lethal effect can be generated, while long-term administration can also generate drug resistance.
Theoretically using chemotherapy and light power link treatment mode, breast cancer can be more thoroughly treated.In addition, by a variety of It is medication combined collaboration medication also by be effective treatment of cancer direction.Optical dynamic therapy and chemotherapy do not have to be mutually exclusive simultaneously, and have There is certain synergistic effect, is conducive to the therapeutic effect for improving breast cancer, therefore there is special status in oncotherapy.For cream The research of gland cancer targeted chemotherapy light power link treatment is in the ascendant, has established theoretical base for effective Therapy study of breast cancer Plinth.But the combination therapy of lipid peroxidation, chemotherapy and optical dynamic therapy based on unsaturated fatty acid, but has not been reported.
Omega-3 unsaturated fatty acid content in deep sea fish oil is plentiful, long-term to take in omega-3 polyunsaturated fatty acid It can reduce the risk with breast cancer.Omega-3 unsaturated fatty acid can be such that breast cancer cell inner lipid Peroxidation Product increases Add, damaging cells film.These peroxide can cause tumour cell internal protein, DN A and lipid membrane disruption, further damage Tumour cell generates free radicals, and makes cell death.The free radical of generation continues and the rouge on other cell membranes, mitochondrial membrane Fat acid chain reaction generates cascade lethal effect to tumour cell.Omega-3 unsaturated fatty acid ancillary drug treats mammary gland simultaneously Cancer, which has been displayed, has preferable collaboration anti-breast cancer effect.
Polyethylene glycol PEG is to obtain the non-ionic water-soluble polymer of food and drug administration's approval, is had Can degrade in vivo, the water solubility of nontoxicity, no antigen, height the advantages that.PEG is by improving nano-medicament carrier surface hydrophilic Property, increase steric hindrance, thus on the one hand make nanoparticle have good stability without assemble, on the other hand protect nanoparticle Not by the phagocytosis of opsonin identification, reduction reticuloendothelial system in blood, to extend the circulation time of nanoparticle in vivo. Most importantly, PEG modification can inhibit immune response generation and can be by its hydrophily, flexibility, biocompatibility etc. Advantageous property is imparted on the monounsaturated fatty acid molecule after modification.
Summary of the invention
It is a kind of based on unsaturated fatty acid nanoparticle the purpose of the invention is to overcome the deficiencies of the prior art and provide The preparation and its application of tumor-targeting drug.
The first purpose of the invention is to provide a kind of micella mPEG-DHA.
A second object of the present invention is to provide the preparation methods of the micella mPEG-DHA.
Third object of the present invention is to provide a kind of micella DSPE-PEG-Anti-HER2。
Fourth object of the present invention also provides the micella DSPE-PEG-Anti-HER2Preparation method.
Fifth object of the present invention is to provide a kind of for lipid peroxidation, light power-chemotherapy combined treatment nanometer Particle.
6th purpose of the invention also provides the preparation method of the nanoparticle.
7th purpose of the invention also provides the micella mPEG-DHA, the micella DSPE-PEG- Anti-HER2 And/or one or more of described nanoparticle is preparing lipid peroxidation, light power-chemotherapy combined treatment HER2Positive carcinoma Application in the drug of disease.
To achieve the goals above, the present invention is achieved by the following technical programs:
Tumor tissues have the following characteristics that cell growth rapidly compared with normal tissue, and oxygen demand increases, in tumor tissues Sensitive targeting effect that is acid, therefore can use this feature realization pH is presented 6.5 or so for pH value；Tumour cell can be with Certain specific receptors are over-expressed, and then express lower in normal tissue, utilize ANTI-HER2And its receptor-specific knot It closes, realizes the active targeting effect of tumor tissues；Tumor tissue growth is out of control, vascular system developmental defect, between capillary Gap increases, and permeability increases, while lacking effective lymphatic return system, " the infiltration of enhancing of the nanoparticle in tumor tissues Property and anelasticity effect " should be the inherent characteristic of nano-carrier, be the theoretical basis of its tumour passive targeting.
Using EDC/NHS activation unsaturated fatty acid (DHA) on carboxyl so that with amino-polyethyleneglycols (m PEG-NH2) Amino carry out covalent bonding reaction, obtain amphipathic chain mPEG-DHA, then utilize amino and carboxyl dehydration, Anti-HER2It modifies on DSPE-PEG chain, obtains targeted molecular chain D SPE-PEG-Anti-HER2, finally by mPEG-DHA, DSPE-PEG-Anti-HER2, photosensitizer M PPa (Methyl Pyropheophorbide) and DOX (adriamycin) mixing, be prepared Tumor-targeting drug DPSPAH/MPPa/DOX based on unsaturated fatty acid nanoparticle.
By characterization methods such as infrared spectroscopy, UV absorption, partial size inspection, transmission electron microscopes, show based on unsaturated fat The drug-loaded nanoparticles of acid successfully synthesize.Pass through MTT detection, DAPI detection, active oxygen and Ca2+Detection, flow cytometry etc. are thin Born of the same parents' experimental verification DPSPAH/MPPa/DOX inhibits the effect of breast cancer cell growth, has in breast cancer cell model and significantly controls Therapeutic effect.
Because of claimed the following contents:
A kind of micella mPEG-DHA, docosahexaenoic acid are connected with amino-polyethyleneglycols.
The amino of the preparation method of the micella mPEG-DHA, the carboxyl of docosahexaenoic acid and amino-polyethyleneglycols into Row condensation reaction.
Preferably, comprising the following steps:
S11. with the carboxyl of EDC/NHS activation docosahexaenoic acid；
S12. amino-polyethyleneglycols are added to be reacted.
Preferably, in step S11, dimethyl sulfoxide (DMSO) solution and dimethyl formyl of dodecahexaene acid (DHA) The N of amine, ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-dimethylformamide (DMF) solution and hydroxyl The N of base thiosuccimide (NHS), solution is sufficiently mixed reaction in N-dimethylformamide (DMF).
Preferably, in step S11, the volume ratio of dimethyl sulfoxide and dimethylformamide is 1:2~5.
More there is a choosing, in step S11, the volume ratio of dimethyl sulfoxide and dimethylformamide is 1:3.
Preferably, in step S11,4~6h of stirring is sufficiently mixed reaction.
It is highly preferred that stirring 4h is sufficiently mixed reaction in step S11.
Preferably, in step S12, amino-polyethyleneglycols are added, are sufficiently mixed reaction, purify drying.
Preferably, in step S12,20~30h of stirring is sufficiently mixed reaction.
It is highly preferred that stirring is sufficiently mixed reaction for 24 hours in step S12.
Preferably, in step S12, pH7.4 PBS dialysis purification.
Preferably, in step S12, freeze-drying.
It is highly preferred that EDC:NHS=1:0.5~5 (mol:mol).
It is further preferred that EDC:NHS=1:1 (mol:mol).
It is highly preferred that DHA:mPEG-NH2=1:1~3 (mol:mol).
It is further preferred that DHA:mPEG-NH2=1:2 (mol:mol).
Most preferably, the preparation method of the micella mPEG-DHA, comprising the following steps:
S11. dimethyl sulfoxide (DMSO) solution of docosahexaenoic acid (DHA) and dimethylformamide, ethyl-(3- Dimethylaminopropyl) carbodiimide hydrochloride (EDC) N, N-dimethylformamide (DMF) solution and hydroxy amber Solution is sufficiently mixed reaction in the n,N dimethylformamide (DMF) of acid imide (NHS), and 4~6h of stirring is sufficiently mixed reaction；
S12. amino-polyethyleneglycols are added to be reacted, 20~30h of stirring is sufficiently mixed reaction, PBS (pH7.4) dialysis Purifying, freeze-drying；
Wherein, EDC:NHS=1:0.5~5 (mol:mol), DHA:mPEG-NH2=1:1~3 (mol:mol), DHA: The volume ratio of EDC:NHS=0.1~0.3:1:0.5~5 (mol:mol), dimethyl sulfoxide and dimethylformamide is 1:2~5.
It is further preferred that the preparation method of the micella mPEG-DHA, comprising the following steps:
S11. dimethyl sulfoxide (DMSO) solution and dimethylformamide, ethyl-(3- bis- of dodecahexaene sour (DHA) Dimethylaminopropyl) carbodiimide hydrochloride (EDC) N, N-dimethylformamide (DMF) solution and hydroxy succinyl Solution is sufficiently mixed reaction in the n,N dimethylformamide (DMF) of imines (NHS), and stirring 4h is sufficiently mixed reaction；
S12. amino-polyethyleneglycols are added to be reacted, stirs and is sufficiently mixed reaction for 24 hours, PBS (pH7.4) dialysis purification, Freeze-drying；
Wherein, EDC:NHS=1:1 (mol:mol), DHA:mPEG-NH2=1:2 (mol:mol), DHA:EDC:NHS= The volume ratio of 0.2:1:1 (mol:mol), dimethyl sulfoxide and dimethylformamide is 1:3.
The micella mPEG-DHA that method made above is prepared, also belongs to protection scope of the present invention.
A kind of micella DSPE-PEG-Anti-HER2, it is connected with Anti-HER2DSPE-PEG.
The micella DSPE-PEG-Anti-HER2Preparation method, the carboxyl of DSPE-PEG-COOH, with Anti-HER2's Amino carries out condensation reaction.
Preferably, comprising the following steps:
S21. with the carboxyl of EDC/NHS activation DSPE-PEG-COOH；
S22. Anti-HER is added2It is reacted.
Preferably, in step S21, ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), hydroxyl sulphur It is dissolved in PBS (pH7.4) for succinimide (NHS) and DSPE-PEG-COOH, is sufficiently mixed reaction.
Preferably, in step S21,4~6h of stirring is sufficiently mixed reaction.
It is highly preferred that stirring 4h is sufficiently mixed reaction in step S21.
Preferably, in step S22, Anti-HER is added2, it is sufficiently mixed reaction, purifies drying.
Preferably, in step S22,20~30h of stirring is sufficiently mixed reaction.
It is highly preferred that stirring is sufficiently mixed reaction for 24 hours in step S22.
Preferably, in step S22, PBS (pH7.4) dialysis purification.
It is highly preferred that in step S22,4 DEG C of PBS (pH7.4) dialysis purifications
Preferably, in step S22, freeze-drying.
It is highly preferred that EDC:NHS=1:0.5~5 (mol:mol).
It is further preferred that EDC:NHS=1:1 (mol:mol).
It is highly preferred that DSPE-PEG-COOH:Anti-HER2=1:1 × 10-6~1 × 10-5(mol:mol).
It is further preferred that DSPE-PEG-COOH:Anti-HER2=1:5.4 × 10-6(mol:mol).
Most preferably, the micella DSPE-PEG-Anti-HER2Preparation method, comprising the following steps:
S21. ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), hydroxy thiosuccinimide (NHS) it is dissolved in PBS (pH7.4) with DSPE-PEG-COOH, EDC/NHS activates the carboxyl of DSPE-PEG-COOH, stirring 4 ~6h is sufficiently mixed reaction；
S22. Anti-HER is added2It is reacted, 20~30h of stirring is sufficiently mixed reaction, and PBS (pH7.4) dialysis is pure Change, freeze-drying；
Wherein, EDC:NHS=1:0.5~5 (mol:mol), DSPE-PEG-COOH:Anti-HER2=1:1 × 10-6~1 ×10-5(mol:mol).
It is further preferred that the micella DSPE-PEG-Anti-HER2Preparation method, comprising the following steps:
S21. ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), hydroxy thiosuccinimide (NHS) it is dissolved in PBS (pH7.4) with DSPE-PEG-COOH, EDC/NHS activates the carboxyl of DSPE-PEG-COOH, stirs 4h It is sufficiently mixed reaction；
S22. Anti-HER is added2It is reacted, stirs and be sufficiently mixed reaction for 24 hours, 4 DEG C of PBS (pH7.4) dialysis purifications, Freeze-drying；
Wherein, EDC:NHS=1:1 (mol:mol), DSPE-PEG-COOH:Anti-HER2=1:5.4 × 10-6(mol: mol)。
The micella DSPE-PEG-Anti-HER that method made above is prepared2, also belong to protection scope of the present invention.
A kind of nanoparticle treated for lipid peroxidation, light power-chemotherapy combined, contains the micella mPEG- DHA, the micella DSPE-PEG-Anti-HER2, Methyl Pyropheophorbide and adriamycin.
The preparation method of the nanoparticle, by the micella mPEG-DHA, the micella DSPE-PEG- Anti- HER2, the mixing in PBS (pH7.4) of Methyl Pyropheophorbide and adriamycin, carry out self assembly to get.
Preferably, comprising the following steps:
S31. by the micella mPEG-DHA, the micella DSPE-PEG-Anti-HER2, Methyl Pyropheophorbide The mixing mixing in PBS (pH7.4) with adriamycin；
S32. it purifies, it is dry.
Preferably, the micella mPEG-DHA, the micella DSPE-PEG-Anti-HER2, Methyl Pyropheophorbide Dosage with adriamycin is 5:1~3:0.1~0.5:0.5~2 in mass.
It is highly preferred that the mPEG-DHA, the DSPE-PEG-Anti-HER2, Methyl Pyropheophorbide and Ah The dosage of mycin is 5:2:0.2:1 in mass.
Preferably, it in step S32, is purified using bag filter
It is highly preferred that the use of specification being that the bag filter that molecular cut off is 1000~2500 carries out dialysis purification.
It is highly preferred that the use of specification being that the bag filter that molecular cut off is 1000 carries out dialysis purification.
Preferably, in step S32, freeze-drying.
Most preferably, the preparation method of the nanoparticle, comprising the following steps:
S31. by the micella mPEG-DHA, the micella DSPE-PEG-Anti-HER2, Methyl Pyropheophorbide With adriamycin hybrid reaction in water, the micella mPEG-DHA, the micella DSPE-PEG-Anti-HER2, burnt de-magging leaf The dosage of green acids-a methyl esters and adriamycin is 5:1~3:0.1~0.5:0.5~2 in mass.
It S32. the use of specification is that the bag filter that molecular cut off is 1000~2500 carries out dialysis purification, freeze-drying.
It is further preferred that the preparation method of the nanoparticle, comprising the following steps:
S31. by the micella mPEG-DHA, the micella DSPE-PEG-Anti-HER2, Methyl Pyropheophorbide With adriamycin hybrid reaction in water, the micella mPEG-DHA, the micella DSPE-PEG-Anti-HER2, burnt de-magging leaf The dosage of green acids-a methyl esters and adriamycin is 5:2:0.2:1 in mass.
It S32. the use of specification is that the bag filter that molecular cut off is 1000 carries out dialysis purification, freeze-drying.
The nanoparticle that method made above is prepared, also belongs to protection scope of the present invention.
The micella mPEG-DHA, the micella DSPE-PEG-Anti-HER2And/or one of described nanoparticle Or it is several in the application prepared in lipid peroxidation, light power-chemotherapy combined treatment HER2 positive cancer drug.
Preferably, the cancer is breast cancer.
The present invention is with breast cancer cell surface HER2For target, using unsaturated fatty acid-polyethylene glycol nano-micelle as light Quick dose and chemotherapeutics carrier, with Anti-HER2For targeted molecular, it is prepared for a kind of novel based on unsaturated fatty acid nanometer The tumor-targeting drug of grain, the advantage of chemotherapy and optical dynamic therapy and lipid peroxidation is combined, mammary gland is overcome The deficiency of cancer monotherapy reaches and improves the target that breast cancer is effectively treated.Pass through the targeting Anti-HER of carrier surface2With it is swollen Oncocyte surface HER2Specific binding realize the selective active targeting tumor locus of Nano medication, assign nanoparticle master Dynamic target function；It is assisted with unsaturated fatty acid hydrophobic effect and carries photosensitizer and chemotherapeutics, while utilizing unsaturated fatty acid Lipid peroxidation assigns Nano medication control-release function in release chemotherapeutics intracellular.Even if non-target tissue accidentally receive by intake Rice drug will not cause optical dynamic therapy, limit nanoparticle release adriamycin, can reduce due to being not affected by light excitation The toxic side effect of DPSPAH/MPPa/DOX embodies the excellent of double target functions of the nanoparticle based on active targeting and light selectivity Gesture helps to realize DPSPAH/MPPa/DOX high-efficiency low-toxicity function.
Compared with prior art, the invention has the following beneficial effects:
A kind of joining for lipid peroxidation, light power-chemotherapy based on unsaturated fatty acid is successfully prepared in the present invention Treatment nanoparticle is closed, for partial size in 190nm or so, stability is good, the release of chemotherapeutics can be effectively controlled, and has good life Object compatibility can utilize lipid peroxidation, optical dynamic therapy and chemotherapy synergistic treatment HER2Positive breast cancer.By to HER2 Positive breast cancer cells antitumor action studies have shown that constructed nanoparticle there is significant killing to make breast cancer cell With normal tissue cell low toxicity.Cancer target nanoparticle based on unsaturated fatty acid nanoparticle of the invention is efficiently low Poison, it is with high application prospect in the fields such as nano-medicament carrier and Nano medication oncotherapy.
Detailed description of the invention
Fig. 1 is that the nano drug-carrying compound based on unsaturated fatty acid synthesizes schematic diagram.
Fig. 2 is Amphiphilic micelle mPEG-DHA infrared spectroscopy.
Fig. 3 is sds polyacrylamide gel electrophoresis qualitative detection Anti-HER2The modification of antibody.
Fig. 4 is drug-loaded nanoparticles partial size schematic diagram, scanning electron microscope (SEM) photograph based on unsaturated fatty acid.
Fig. 5 is the characteristic absorption peak of nano-micelle encapsulating medicine.
Fig. 6 is that mPEG-DHA is minimum at micella Concentration Testing.
Fig. 7 is peroxidatic reaction of lipid detection.
Fig. 8 is adriamycin medicine realeasing rate detection under condition of different pH that nano drug-carrying micella contains.
Fig. 9 is different time sections morphological observation after the processing of nano drug-carrying micella.
Figure 10 is detection of the Nano medication to the killing-efficiency of MCF7 cell of various concentration.
Figure 11 is the therapeutic effect that different pharmaceutical processing group life or death cell dyeing detects Nano medication.
Figure 12 is the fluorescence detection of adriamycin under the conditions of different disposal.
Figure 13 is active oxygen and calcium ion detection schematic diagram under different Nano medication treatment conditions.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained And material.
1, cell strain
Human breast cancer cell (MCF-7 cell line) is provided by Shenzhen Institutes of Advanced Technology, Chinese Academy of Science, through this experiment Room secondary culture.
2, main agents
Photosensitizer (MPPa), adriamycin (DOX), docosahexaenoic acid (DHA) are purchased from lark prestige Science and Technology Ltd.； DSPE-PEG-COOH molecular weight 2000 is purchased from Shanghai Ai Weite Pharmaceutical Technology Co., Ltd, Anti-HER2Rabbit pAb purchase Big biology is created from Guangzhou；PEG-NH2Molecular weight 2000 is purchased from Zhen Zhun Reagent Company.
3, instrument
Sigma32184 high speed freezing centrifuge, Thermo CO2Incubator, Medical Instruments factory, Jintan City, Jiangsu Province 78-1 Magnetic stirring apparatus, HV-85 autoclave, aseptic operating platform, Guangzhou Ke Qiao experimental technique equipment Co., Ltd thermostat water bath Deng.
The synthesis of 1 nanoparticle DPSPAH/MPPa/DOX of embodiment
1, the preparation of mPEG-DHA chain.
The carboxyl of unsaturated fatty acid (DHA), and then and amino-polyethyleneglycols are activated in solvent DMF using EDC/NHS (mPEG-NH2) amino occur condensation reaction, formed mPEG-DHA chain.
Specific practice: by 100 μm of ol (19.17mg) ethyl-(3- dimethylaminopropyl) carbodiimide hydrochlorides (EDC) it is dissolved in 1mL dimethylformamide (DMF), 100 μm of ol (11.509mg) hydroxy thiosuccinimide (NHS) dissolutions At 1mL dimethylformamide (DMF), the docosahexaenoic acid (DHA) of 20 μm of ol (6.57mg) first uses the dimethyl sulfoxide of 1mL Then the DMF of 3mL is added in dissolution, later mix DHA lysate and EDC/NHS lysate, stir 4h at room temperature, then plus Enter 40 μm of ol (80mg) mPEG-NH of amino-containing hydrophilic substance2, stir at room temperature for 24 hours.Finally dialyse at PBS (pH7.4), with It is freeze-dried afterwards with freeze dryer, obtains micella mPEG-DHA.
2, micella DSPE-PEG-Anti-HER2Preparation.
Using the condensation reaction of carboxyl and amino Anti-HER2It modifies on DSPE-PEG chain, forms micella DSPE- PEG-Anti-HER2。
Specific practice: by 20 μm of ol (3.714mg) ethyl-(3- dimethylaminopropyl) carbodiimide hydrochlorides (EDC) it is dissolved in 1ml PBS (pH7.4), 20 μm of ol (2.3018mg) N- hydroxy thiosuccinimides (NHS) are dissolved in In 1mL PBS (pH7.4), 4 μm of ol (8mg) DSPE-PEG-COOH are dissolved in 3mL PBS (pH7.4), later by DSPE-PEG Lysate and EDC/NHS lysate mix, and stir 4h in ice-water bath, amino-containing Anti-HER is then added2 4μg(2.16× 10-5μm ol), it stirs for 24 hours in ice-water bath, the finally dialysis in 4 DEG C of PBS (pH7.4) is then freeze-dried with freeze dryer, is obtained Micella DSPE-PEG-Anti-HER2。
3, lipid peroxidation, light power-chemotherapy combined treatment nano drug-carrying that can be used for based on unsaturated fatty acid are answered Close the synthesis of object DPSPAH/MPPa/DOX.
By micella mPEG-DHA and micella DSPE-PEG-Anti-HER2, photosensitizer MPPa (pyropheophorbide-a first Ester), chemotherapeutics DOX (adriamycin) pass through self assembly wrap up photosensitizer and chemotherapeutics lipid peroxidation, light power- The nano medicament carrying system DPSPAH/MPPa/DOX of chemotherapy combined treatment.
Specific practice: precision weighing amphipathic nature material mPEG-DHA (5mg) and DSPE-PEG-Anti-HER2 (2mg) is dredged Aqueous pharmaceutical photosensitizer MPPa (0.2mg) and chemotherapeutic drugs Doxorubicin (1mg) (mPEG-DHA:DSPE-PEG- calculated by mass Anti-HER2: MPPa:DOX=5:2:0.2:1), material and drug are dissolved in 10mL PBS (pH7.4), magnetic agitation makes It mixes.Solution is transferred in the bag filter that molecular cut off is 1000, is placed in 2L deionized water under the conditions of 4 DEG C thoroughly again Analysis, respectively at 2h, 4h, 6h, 8h, 12h, replaces fresh deionized water for 24 hours.After the completion of dialysis, the solution in bag filter is taken out, It is centrifuged 10min with 3000rmin, for supernatant via 0.22 μm of filtering with microporous membrane, gained nano-particle solution is put in 4 DEG C of ice It is stand-by in case.
Synthetic route is as shown in Figure 1.
The synthesis of the non-medicine-carried nano particles DSSPAH of reference examples
By the micella mPEG-DHA being prepared in embodiment 1 and the micella DSPE-PEG- being prepared in embodiment 1 Anti-HER2Pass through self assembly medicine nanoparticle DSSPAH.Material and drug are dissolved in 10mL PBS (pH7.4), Magnetic agitation makes to mix.
Specific practice: precision weighing amphipathic nature material mPEG-DHA (5mg) and DSPE-PEG-Anti-HER2(2mg), will Material is dissolved in 10mL PBS (pH7.4), and magnetic agitation makes to mix.The non-medicine-carried nano particles DSSPAH solution of gained is put in 4 It is stand-by in DEG C refrigerator.
2 micella mPEG-DHA of embodiment, micella DSPE-PEG-Anti-HER2With the characterization of nanoparticle
One, infrared spectroscopy detects
1, experimental method
In infrared inspection process, we predominantly detect the formation for whether having amido bond.Specific detection method: by what is prepared Solid is made with vacuum freeze drier in micella mPEG-DHA, takes 3mg or so, separately take respectively 3mg or so amino PEG and DHA.Sample is had to 40 DEG C of baking ovens and is dried before infrared detection.Dried sample and KBr are done in drying machine 1~2mg sample to be tested is mixed with the pure KBr of 200mg and is ground uniformly, and mixture is ground to granularity less than 2 μ by dry processing M, to avoid stray light effects.Mixture is placed in mold, is pressed into mixture with 5~10MPa pressure on hydraulic press Bright thin slice, upper machine measurement.
2, experimental result
As a result as shown in Fig. 2, the carboxyl characteristic absorption peak 1639 (C=0) of unsaturated fatty acid, (3404) OH, amino 3370 (the NH of carboxyl characteristic absorption peak of mPEG2), all successfully it detected.According to the infrared detection to micella mPEG-DHA, knot Fruit shows that the corresponding correlation absorption band (N-H/3412, C=O/1656) of amido bond is all found in infrared results, therefore successfully Prove that amino and carboxyl form amido bond, the amino of the carboxyl and amino mPEG that illustrate unsaturated fatty acid DHA successfully reacts shape At amido bond, micella mPEG-DHA is formed.
Two, sds polyacrylamide gel electrophoresis qualitative characterization nano drug-carrying micelle complex
1, experimental method
In order to further prove Anti-HER2Success is modified onto amphipathic chain DSPE-PEG-COOH, by Anti-HER2 And Anti-HER2The nano drug-carrying micella of modified carries out one polyacrylamide gels (SDS- of lauryl sodium sulfate PAGE) qualitative characterization.SDS-PAGE Coomassie brilliant blue protein staining be using dyeing liquor and albumen by Van der Waals force in conjunction with, it is suitable Micro dyeing for albumen.
2, experimental result
As a result as shown in figure 3, band is from left to right respectively albumen Marker (M), Anti-HER2It is prepared with embodiment 2 Nano-micelle (DPSPAH).By SDS-PAGE protein staining it may be seen that in molecular weight 170KDa or so, Wo Menke Significantly to observe simple Anti-HER2Group has band, and DPSPAH group also has band, can demonstrate,prove Anti-HER2Successfully It modifies on DPSPAH.
Three, droplet measurement
1, experimental method
Whether droplet measurement forms for preliminary judgement nanosized micelles, and transmission electron microscope detection is in order to apparent intuitive The pattern of synthesizing nano-particle is observed, and determines whether to coincide with droplet measurement data.
Specific practice: the nano drug-carrying compound (DPSPAH/MPPa/ for taking the embodiment 1 of 300 μ L or so to be prepared DOX droplet measurement) is carried out.Sample to be measured shaking table is placed on before droplet measurement to divide with per minute 100 or so frequency oscillation 20 Clock.Nano particle diameter detection is measured using nanoanalysis instrument.In particle size determination, sample is dissolved in PBS, minimum to measure 180s.Instrument is multiple by automatic measurement, and result is obtained hydrodynamics particle size results according to correlation analysis.
2, experimental result
As a result as shown in figure 4, the nanoparticle for containing chemotherapeutic drugs Doxorubicin and photosensitizer that display embodiment 1 is prepared Sub- DPSPAH/MPPa/DOX particle size is 190nm or so.
Four, transmission electron microscope detects.
1, experimental method
The suspension for the nanoparticle DPSPAH/MPPa/DOX that embodiment 1 is prepared drips on the copper mesh for being covered with carbon film, 5min is stood, excessive moisture is blotted with filter paper, stands 30min, parched completely to it.Using 7650 transmission electron microscope of HITACHI, Electron accelerating voltage is 300kV, respectively at 1 × 104Again, 2 × 104Under amplification factor again, particle shape is observed.
2, experimental result
As a result as shown in figure 4, Electronic Speculum result further displays the nanoparticle DPSPAH/ that embodiment 1 is finally made MPPa/DOX, particle size is 190nm or so, spherical, and dispersibility preferably, reaches expected experiment purpose.And the particle size Nano medication can carry out the various physicochemical characterizations of next step and external cell experiment and intracorporal zoopery are carried The effect of medicine nanoparticle is confirmed.
Five, UV absorbance detection
1, experimental method
Take the DOX of 1mg to be dissolved in the DMSO of 10mL, after completely dissolution prepare 0.1mg/mL, 0.05mg/mL, The solution of the various concentrations such as 0.025mg/mL, 0.0125mg/mL, 0.00625mg/mL surveys ultraviolet suction with ultraviolet specrophotometer It receives, the section of measurement is 300~600nm, before surveying UV absorption, by sample to be tested ultrasound 2 minutes, to guarantee that solute is sufficiently molten Solution.The UV absorption of same method measurement photosensitizer MPPa, the range of measurement is 500-800nm.In final nano drug-carrying The nano-micelle synthesized after particle synthesis to DOX, MPPa and finally carries out determination of uv absorption.
2, experimental result
UV absorbance detection is carried out to chemotherapeutics DOX, photosensitizer MPPa and the nano drug-carrying micella finally synthesized respectively, The characteristic absorption peak of (Fig. 5) DOX is at 490nm as the result is shown, and the characteristic absorption peak of the MPPa of photosensitizer is at 668nm, and most Also there are the two characteristic absorption peaks in the nano drug-carrying micella synthesized eventually, illustrates that two kinds of drugs for treating tumour successfully contain To in nano-micelle.
In order to quantitative determine the amount of area load DOX on the nanoparticle finally synthesized, first with UV absorption light Spectrum determine a series of various concentrations DOX absorption spectrum, using DOX concentration of standard solution as abscissa its corresponding 490nm The absorption intensity at place is ordinate, draws it and absorbs standard curve.It can be seen that good in 5~100 μ g/mL range linear relationships Good, linear regression is carried out to it can obtain its linear equation are as follows: Y=21.141X+0.0457R2=0.9978.
While in order to quantitative determine the amount for the photosensitizer MPPa that the nanoparticle of synthesis wraps up above, first with purple A series of outer absorption spectromtry absorption spectrum of the MPPa of various concentrations, using MPPa concentration of standard solution as abscissa, Absorption intensity at corresponding 668nm is ordinate, draws it and absorbs standard curve.It can be seen that linear in 5~80 μM of ranges Relationship is good, its linear equation can be obtained by carrying out linear regression to it are as follows: Y=0.0357X+0.0035R2=0.9999.
Six, conductivity detects
1, experimental method
By mPEG-DHA gradient dilution, conductivity is measured respectively, conductivity is in higher solution concentration and lower solution concentration It is lower linear with solution concentration respectively, and it is minimum at the generation of micellar concentration (CMC) conductivity near critical micelle concentration There is an apparent inflection point, calculates this inflection point according to two straight lines in significant change, it can at a temperature of obtaining this CMC。
2, experimental result
As a result as shown in fig. 6, the conductivity of mPEG-DHA changes at 0.42mg/mL, therefore mPEG-DHA CMC is 0.42mg/mL.
Seven, mda content detects
1, experimental method
In order to which peroxidatic reaction of lipid can occur for quantitative detection unsaturated fatty acid DHA, we use malonaldehyde (MDA) Testing cassete is detected.
2, experimental result
As a result as shown in fig. 7, unsaturated fatty acid (DHA) and photosensitizer MPPa generate the third two under laser irradiation condition Aldehyde, malonaldehyde are the product of lipid oxidation reaction, therefore illustrate that peroxidatic reaction of lipid can occur for DHA.On a cellular level, Lipid peroxidation is the process of the ROS oxidative biological film occurred after oxidative stress enhances, i.e. phosphatide, enzyme and the film of ROS and biomembrane The macromolecular substances such as the side chain and nucleic acid of the relevant polyunsaturated fatty acid of receptor play peroxidatic reaction of lipid and form lipid peroxy Change product, such as malonaldehyde (MDA) and 4- hydroxyl nonenoic acid (HNE), wherein MDA is lipid peroxidation and cell oxidative damage An important Testing index.
Eight, encapsulation rate detects
1, experimental method
The carrier micelle DPSPAH//MPPa/DOX for containing drug DOX and photosensitizer MPPa is freeze-dried into powder.It takes The nanoparticle of 1mg synthesis is dissolved into suspension with DMSO.Its drug absorption spectrum, and foundation are measured using ultraviolet specrophotometer Its light absorption value conversion medicament contg at the wavelength for drawing standard curve.The encapsulation rate of drug calculates according to the following formula:
Encapsulation rate (EE)=(medication amount/dosage in nano drug-carrying example) × 100%；
2, experimental result
Being calculated according to the standard curve measured above and being computed the encapsulation rate of DOX is 56.65%；The encapsulating of MPPA Rate is 52.71%.
Nine, medicine realeasing rate detects
1, experimental method
By the nano drug-carrying micella DPSPAH//MPPa/DOX of synthesis, under the conditions of 37 DEG C with respectively with pH7.4 PBS and The PBS of pH6.5 is divided into four groups under the conditions of irradiating laser (660nm) and being protected from light two kinds and dialyses, incipient stage drug release Comparatively fast, a sample is taken every a hour, sample time is appropriately extended later in 9 hours, and the sample taken is surveyed UV absorption, root According to the standard curve done before, the drug release rate of DOX is calculated.We calculate the calculation formula of DOX drug release are as follows:
Medicine realeasing rate=(medication amount/micella contains medication amount in dialyzate PBS) × 100%；
2, experimental result
As shown in figure 8, respectively under conditions of pH value is 6.4 and 7.4, pH6.4 adds under the conditions of laser irradiation group at 37 DEG C Faster, and the drug release rate under the conditions of final pH 6.4 plus laser irradiation group is also obviously up to obvious drug release 92.21%, and under the conditions of pH7.4 relatively low only 58.03% laser irradiation drug release rate.Therefore can speculate Our drug cooperates external laser irradiation under the conditions of tumor microenvironment, and the effect of control release may be implemented, and to pH The normal tissue being in neutrality has smaller toxic side effect.
3 nanoparticle of embodiment inhibits breast cancer in vitro
One, morphological observation
1, experimental method
Cultured cell is taken out from incubator, pancreatin digests 2min, and 4mL culture solution is added to blow and beat cell.Take 3mL cell Suspension is added in centrifuge tube, adds 1mL culture solution, blows and beats cell, so that cell is suspended uniform.It is outstanding that 100 μ L cells are drawn with liquid-transfering gun Liquid is added in 24 orifice plates, makes cell adhere-wall culture 6h.The nano drug-carrying micella of synthesis is taken out under the conditions of being protected from light, slightly concussion is shaken It is even.Cultured cell is taken out, cell waste liquid is sucked, PBS is added to wash twice, fresh culture is added.Addition embodiment 1 is made later Standby obtained nanoparticle DPSPAH/MPPa/DOX, is protected from light and is incubated for 4h, according to laser (660nm, 100mW/cm2, 10min). 0h, 6h, 12h are cultivated in the incubator and clap cellular morphology photo for 24 hours.
2, experimental result
Experimental result is as shown in Figure 9, it can be clearly seen that as apparent change occurs for drug treating time cellular morphology, Cellular morphology is substantially all when acting on 6h becomes round, and starts a large amount of phenomena of apoptosis occur, after effect for 24 hours, cell base This becomes cell fragment.The nanoparticle that can be prepared by setting aside morphological observation with preliminary judgement embodiment 1 DPSPAH/MPPa/DOX is that have apparent fragmentation effect to cell.
Two, cell killing Efficiency testing
1, experimental method
(1) the nano drug-carrying micella killing-efficiency of MTT detection synthesis
Cultured cell is taken out from incubator, pancreatin digests 2min, and 4mL culture solution is added to blow and beat cell.Take 2mL cell Suspension is added in centrifuge tube, adds 3mL culture solution, blows and beats cell, so that cell is suspended uniform.It is outstanding that 100 μ L cells are drawn with liquid-transfering gun Liquid is added in 96 orifice plates, makes cell adhere-wall culture 6h.Cultured cell is taken out, cell waste liquid is sucked, adds PBS to wash twice, adds Enter fresh culture.The nanoparticle DPSPAH/MPPa/DOX that addition embodiment 1 is prepared later, is protected from light and is incubated for 4h, shines Laser (660nm, 100mW/cm2, 10min) and shift to an earlier date 4h, MTT10 μ L is added, after being incubated for 4h, liquid-transfering gun is drawn waste liquid and (paid attention to not Wash bottom hole crystal), then 150 μ L DMSO are added in every hole, dissolve bottom hole crystal, and (attention is protected from light) 550nm surveys OD value.Make Data are handled with SPSS data processing software, PS does figure.
(2) the bis- dyes of AO/PI
Cultured cell is taken out from incubator, pancreatin digests 2min, and 4mL culture solution is added to blow and beat cell.Take 2mL cell Suspension is added in centrifuge tube, and 8mL culture solution is added to blow and beat cell, so that cell is suspended uniform.1mL cell suspension is drawn with liquid-transfering gun to add Into 24 orifice plates, make cell adhere-wall culture 6h.Cultured cell is taken out, cell waste liquid is sucked, PBS is added to wash twice, is added new Fresh culture medium.The nanoparticle DPSPAH/MPPa/DOX that addition embodiment 1 is prepared later, is protected from light and is incubated for 4h, irradiates laser (660nm, 100mW/cm2, 10min).Waste liquid is drawn, rinses cell twice using PBS.AO/PI dye liquor is added and is incubated for 20 points Waste liquid is quickly sucked out in clock, and PBS is added and washes twice, takes pictures under fluorescence microscope.Data are handled using Photoshop.
(3) DAPI is dyed
Cultured cell is taken out from incubator, pancreatin digests 2min, and 4mL culture solution is added to blow and beat cell.Take 3mL cell Suspension is added in centrifuge tube, adds 11mL culture solution, blows and beats cell, so that cell is suspended uniform.1mL cell suspension is drawn with liquid-transfering gun It is added in 24 orifice plates, makes cell adhere-wall culture 6h.Cultured cell is taken out, cell waste liquid is sucked, PBS is added to wash twice, is added Fresh culture.The nano drug-carrying micella (DPSPAH/MPPa/DOX) for adding synthesis later is protected from light and is incubated for 4h, irradiates laser (660nm, 100mW/cm2, 10min).It cultivates in the incubator for 24 hours, draws waste liquid, PBS is washed twice.Add configured DAPI Dyeing liquor dyes 10min, draws waste liquid, and PBS is washed twice.It is taken pictures using fluorescence microscope, PS handles picture.
2, experimental result
Cell killing efficiency is to detect an important indicator of drug effectiveness.As shown in Figure 10, not with MTT measurement With treated cell viability.Firstly, breast carcinoma cell strain MCF7 is handled with DPSPAH, cell viability is measured, is received as the result is shown Apparent cytotoxicity is not observed in rice corpuscles under a high concentration condition, shows the nano-micelle carrier for not carrying medicine With good biocompatibility.Next, being handled with the nanoparticle DPSPAH/MPPa/DOX that embodiment 1 is prepared thin Born of the same parents, as shown, the activity of cell is also obviously inhibited under the conditions of being protected from light, and with the raising of drug concentration, to cell Lethal effect enhancing, and under conditions of irradiating laser, compared to being protected from light group, work of the nano drug-carrying micella of comparable sodium to cell Property inhibit it is more obvious, as the result is shown photosensitizer (MPPa) more can significantly cooperate with chemotherapeutics (DOX) enhancing nanometer carry Lethal effect of the medicine micella to breast cancer cell.
As shown in figure 11, we identify the life or death cell under different pharmaceutical treatment conditions with the bis- dyes of AO/PI, can be more straight The nanoparticle DPSPAH/MPPa/DOX that the observation embodiment 1 of sight is prepared is at different conditions to the lethal effect of cell. Unloaded nano-micelle DPSPAH is shown in the case where being protected from light and irradiating lasing condition to cell without apparent toxic side effect as the result is shown Show the good biocompatibility of nano drug-carrying micella, and DPSPAH/MPPa/DOX group is compared in the case where irradiating lasing condition and is protected from light Ancestral has more obvious lethal effect to cell.As shown in figure 12, DAPI dyestuff is a kind of fluorescence dye that can be combined with DNA strength Material, because DAPI can penetrate complete cell membrane, it can be used for the dyeing of living cells and fixed cell, and DOX is glimmering It, can be relatively more straight it can be observed that red fluorescence, we are detected by DAPI blue-fluorescence and DOX red fluorescence under light microscope The successful penetrating cell film of nano drug-carrying micella for finding out that we synthesize seen enters and plays therapeutic effect in tumour cell.
Three, active oxygen detection kit detection ROS release
1, experimental method
Cultured cell is taken out from incubator, pancreatin digests 2min, and 4mL culture solution is added to blow and beat cell.Take 2mL cell Suspension is added in centrifuge tube, adds 3mL culture solution, blows and beats cell, so that cell is suspended uniform.Liquid-transfering gun draws 100 μ L cell suspensions It is added in 96 orifice plates, makes cell adhere-wall culture 6h.Cultured cell is taken out, cell waste liquid is sucked, PBS is added to wash twice, is added Fresh culture.The nanoparticle DPSPAH/MPPa/DOX that addition embodiment 1 is prepared later, is protected from light and is incubated for 4h, and irradiation swashs Light (660nm, 100mW/cm2, 10min).0.1 μ L of DCFH-DA is added in 45min in advance, and after being incubated for 45min, PBS washing is thin Born of the same parents 2 times.It is taken pictures using fluorescence microscope, PS does map analysis.
2, experimental result
DCFH-DA can pass freely through cell membrane, into after intracellular, can be generated by intracellular esterase hydrolyzed DCFH.And DCFH is unable to permeabilized cells film, so that DCFH probe be made to be loaded into the cell.Intracellular active oxygen can aoxidize Non-blooming DCFH generates the DCF for having fluorescence.Detect DCF fluorescence it is known that reactive oxygen species level.Such as Figure 13 It is shown, under the particle action condition of same concentrations, photosensitizer MPPa is added and conspicuousness occurs for irradiation laser, activity keto concentration Rising, display nanoparticle can pass through stimulating activity oxygen (optical dynamic therapy), adriamycin chemotherapy and lipid peroxidation killing Cell.
Four, Fluo-3 AM detects calcium ion concentration
1, experimental method
Cultured cell is taken out from incubator, pancreatin digests 2min, and 4mL culture solution is added to blow and beat cell.Take 2mL cell Suspension is added in centrifuge tube, adds 3mL culture solution, blows and beats cell, so that cell is suspended uniform.It is outstanding that 100 μ L cells are drawn with liquid-transfering gun Liquid is added in 96 orifice plates, makes cell adhere-wall culture 6h.Cultured cell is taken out, cell waste liquid is sucked, adds PBS to wash twice, adds Enter fresh culture.The nanoparticle DPSPAH/MPPa/DOX that addition embodiment 1 is prepared later, is protected from light and is incubated for 4h, shines Penetrate laser (660nm, 100mW/cm2, 10min).Waste liquid is drawn, rinses cell twice using PBS.45min in advance is added The Fluo-3 AM fluorescent dye of 0.1 μ L, after being incubated for 45min, PBS is washed cell 2 times, and serum free medium is added.It uses 488nm excitation wavelength, 525nm launch wavelength survey OD value.Data are handled using SPSS data processing software, PS does figure.
2, experimental result
Calcium ion plays an important role in the physiological activity of cell, the variation of free calcium ion concentration and the function of cell Energy, the transmitting of signal and damage and apoptosis suffer from close connection.Calcium ion is individual cells existence and dead signal.Together When, the variation of intracellular free calcium level is also one of the research means of Apoptosis.Fluo-3 AM is that one kind can be with penetrating cell Film detects intracellular Ca2+The fluorescent dye of concentration variation.In the later period of Apoptosis, calcium channel open, intracellular Ca2+ from Sub- concentration increases, and passes through the process of different mode finely regulating apoptosis.As shown in figure 13, simple photosensitizer group is being protected from light item The fluorescence intensity of calcium ion is without significant change under part, and the enhancing of calcium ion fluorescence intensity is more obvious after irradiating laser, and carries Photosensitizer and adriamycin group DPSPAH/MPPa/DOX the calcium ion fluorescence intensity under laser irradiation are remarkably reinforced.Pass through active oxygen With calcium ion detection and mda content detection, illustrate that invented DPSPAH/MPPa/DOX Nano medication has lipid mistake It aoxidizes, the function of chemotherapy and optical dynamic therapy, effectively increases to HER2The killing-efficiency of positive breast cancer cells.

Claims (10)

1. a kind of micella mPEG-DHA, which is characterized in that docosahexaenoic acid is connected with amino-polyethyleneglycols.
2. the preparation method of micella mPEG-DHA described in claim 1, which is characterized in that the carboxyl and ammonia of docosahexaenoic acid The amino of base polyethylene glycol carries out condensation reaction.
3. preparation method according to claim 2, which comprises the following steps:
S11. with the carboxyl of EDC/NHS activation docosahexaenoic acid；
S12. amino-polyethyleneglycols are added to be reacted.
4. a kind of micella DSPE-PEG-Anti-HER2, which is characterized in that it is connected with Anti-HER2DSPE-PEG.
5. micella DSPE-PEG-Anti-HER described in claim 42Preparation method, which is characterized in that DSPE-PEG-COOH's Carboxyl and Anti-HER2Amino carry out condensation reaction.
6. preparation method according to claim 5, which comprises the following steps:
S21. with the carboxyl of EDC/NHS activation DSPE-PEG-COOH；
S22. Anti-HER is added2It is reacted.
7. a kind of for lipid peroxidation, light power-chemotherapy combined treatment nanoparticle, which is characterized in that wanted containing having the right Seek micella DSPE-PEG-Anti-HER described in the 1 micella mPEG-DHA, claim 42, Methyl Pyropheophorbide and Adriamycin.
8. the preparation method of nanoparticle described in claim 5, which is characterized in that by micella mPEG-DHA described in claim 1, Micella DSPE-PEG-Anti-HER described in claim 42, Methyl Pyropheophorbide and adriamycin mixed in PBS, carry out Self assembly to get.
9. preparation method according to claim 8, which comprises the following steps:
S31. by DSPE-PEG-Anti-HER described in mPEG-DHA, claim 4 described in claim 12, pyropheophorbide-a Methyl esters and adriamycin are mixed in PBS；
S32. it purifies, is freeze-dried.
10. micella DSPE-PEG-Anti-HER described in micella mPEG-DHA, claim 4 described in claim 12And/or right It is required that one or more of 7 described nanoparticles are preparing lipid peroxidation, light power-chemotherapy combined treatment HER2Positive carcinoma Application in the drug of disease.
CN201910356708.3A 2019-04-29 2019-04-29 Preparation and application of tumor targeting drug based on unsaturated fatty acid nanoparticles Active CN110314136B (en)

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