CN106511296A - Tumor-targeted self-assembly core-shell drug-loaded nanoparticles preparation method and application thereof - Google Patents
Tumor-targeted self-assembly core-shell drug-loaded nanoparticles preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a tumor-targeted self-assembly core-shell drug-loaded nanoparticles preparation method and an application thereof, which can effectively solve the problems of large toxic and side effect, poor biological compatibility, and poor targeting of the antitumor drug in the prior art. The method comprises the following steps: preparing a polylactic acid-glycolic acid copolymer carboxyl activator and aminated hyaluronic acid, assembling a segmented copolymer, and finally preparing the tumor-targeted self-assembly core-shell drug-loaded nanoparticles. The method has the advantages of simple process, easy operation, abundant raw material source, low cost, small toxic and side effect, and easy realization of industrial production; the prepared tumor-targeted self-assembly core-shell drug-loaded nanoparticles as a drug delivering system has high stability, physical and chemical properties are adjustable; a surface functional group can be used for realizing ligand binding to promote positioning and increase selectivity, targeting release of the drug on a tumor position is urged, so that the sonodynamics and chemotherapy are combined for resisting tumor.
Description
Technical field
The present invention relates to pharmaceutical technology field, the preparation of the hud typed drug-carrying nanometer particle of particularly a kind of cancer target self assembly
Method and application.
Background technology
Nanoparticle (nanopartilcles, NP) be by made by naturally occurring or synthetic macromolecular material particle diameter between 1-1000
Nm solid colloid particles, including nanosphere（) and nanocapsule Nanospheres（Nanocapsules).Active component (medicine, life
Thing active material etc.) can dissolve, be wrapped in inside particles, or adsorb, be attached to particle surface.NP has many merits：（1）
Polymer nanoparticle has higher stability；（2）The controllability of physics and chemical property and biodegradable；（3）Can pass through
Surface reaches targeting purpose by the use of special material, promotes the absorption of medicine and the bioavailability of raising medicine；（4）
Can the existing antitumor drug toxic and side effects of effectively solving it is big, poor biocompatibility, the problem of targeting difference；（5）It is widely used in
Cancer target passs the carrier material of release system.
Sound motivation therapy（Sonodynamic theropy, SDT）The advantage of deep tissues can be penetrated using ultrasound, swashed
Send out targeting and strong antitumor action is produced in the sound sensitiser of tumor locus, have the advantages that side reaction is relatively fewer, because
And to can not perform the operation and tolerate put, change treatment old patient it is particularly suitable, it is traditional especially for those great majority
Perform the operation, put, change fail to respond to any medical treatment or late tumor patient, be a kind of more preferably Therapeutic Method.Ultrasonic in combination sound sensitiser is controlled
The sound motivation therapy (SDT) for treating cancer is suggested with a kind of new anti-tumor method.Sound sensitiser is produced work after ultrasonic activation
Property oxygen can produce strong Graft Versus Tumor, better than the curative effect of simple application ultrasound, it is characterised in that surpassed by sensing high intensity
The biological effect that sound effect is produced to tumor tissues, so as to kill tumor cell or tissue.
Arteannuin is the Sesquiterpene lactones medicine containing peroxy-radical extracted in Chinese medicine Herba Artemisiae annuae, dihydroartemisinine,
Artesunate etc. is the important derivatives of arteannuin.Arteannuin chemical constitution is unique, and antitumous effect is projected, and physiological disposition compares
Stable, individual variation is less, and partly the sad phase is shorter, eliminates very fast, and apparent volume of distribution is larger, and delay is shorter in vivo, is difficult accumulation,
Toxic and side effects are little, cheap, in the treatment of tumor disease, by global affirmative.Once there is document report, in mitochondrion
Interior arteannuin is heme iron or hemoglobin rather than free iron ion effect activation, disturbs mitochondrion electronic chain（ETC) urge
Change peroxide bridge fracture, produce oxygen-derived free radicals first, then Fe2+Reset with peroxide and be converted into carbon radicals, it is thin so as to induce
Born of the same parents' apoptosis.And document was also once reported, arteannuin can cause 1,2,4- trioxane structures to produce as sound sensitiser joint sound motivation therapy
The poisonous active oxygen of life, so as to have certain Proliferation Ability or lethal effect to tumor cell.Therefore, the antitumor of arteannuin
The active oxygen that effect not only can be mediated by heme iron causes apoptosis, but also can be by the effect of extraneous ultrasound
Realize the effect of killing tumor cell.
The premise compound of haemachrome is 5-ALA, on the one hand, the compound can be closed in cell mitochondrial
Into PpIX, PpIX under the catalysis of ferrochelatase with Fe2+Heme is generated, wound is not resulted in human normal cell
Evil.But tumor is with some abnormal cells due to deaminases (porphobilinogen deaminase, PBGD) rising, ferrous iron
Chelatase (ferrochelatase, FC) is reduced, when system or after administer locally to exogenous 5-ALA, normal metabolic balance quilt
Break, 5-ALA is optionally absorbed by tumor cell, generates substantial amounts of PpIX, and be accumulated in tumor cell in its mitochondrion
In, Ultrasound-activated Hematoporphyrin Derivatives, do not affect it is biomembranous under the premise of, endogenous stimulation mitochondrion Capase rely on signal lead to
Road, inducing apoptosis of tumour cell, so as to play lethal effect to sick cell.On the other hand, PpIX urging in ferrochelatase
Change lower and Fe2+Heme is generated, the haemachrome of intracellular can mediate the cytotoxicity of arteannuin, increase haemachrome synthesis
Precursor substance can be remarkably reinforced the antitumor action of arteannuin, therefore haemachrome is the essential condition that arteannuin plays antitumaous effect
One of.
The content of the invention
For above-mentioned situation, the purpose to overcome the defect of prior art, the present invention is just to provide a kind of cancer target certainly
Assemble the preparation method and application of hud typed drug-carrying nanometer particle, can the existing antitumor drug toxic and side effects of effectively solving it is big, it is biological
Poor compatibility, the problem of targeting difference.
The technical scheme of solution is：A kind of preparation method of the hud typed drug-carrying nanometer particle of cancer target self assembly, including with
Lower step：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.1-0.8 g poly lactic-co-glycolic acids altogether
Polymers ultrasonic dissolution adds 42-50 mg N-hydroxy-succinamides and 70-80 mg 1- in 25-30 ml dichloromethane
Ethyl-（3- dimethylaminopropyls）Carbodiimide hydrochloride, magnetic agitation 12-24 h add 2-3 times and mix into mixed liquor
The absolute ether precipitation of the pre-cooling of liquid product, is stored at room temperature 10 min, then the absolute ether with pre-cooling and absolute methanol equal-volume
Mixed liquor rinse precipitate 3 ~ 5 times, be vacuum dried 72 h, obtain Poly(D,L-lactide-co-glycolide activated carboxylic thing；Described
Poly(D,L-lactide-co-glycolide is poly- for end carboxyl PLGA, polylactic acid acetate multipolymer or mono methoxy
One kind in ethylene glycol PLGA；
（2）The preparation of amination hyaluronic acid：0.08-0.15 g hyaluronic acids ultrasound or 50 DEG C of oil baths are weighed, is dissolved completely in
In the Methanamide of 15-20 ml, room temperature is cooled to, adds 500-520 mg EDC and 300-315 mg NHS, 8- is stirred at room temperature
24 h, under ice bath, 10 ml ethylenediamines of Deca and Methanamide are according to volume ratio 1 ~ 4：9 mixed liquor, continues ice bath reaction 0.5-2
H, continues stirring 1-4 h at room temperature into reactant liquor, adds the acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor, crystallize, gained
0.22 μm of organic membrane sucking filtration of crystal obtains filter cake, then with washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h,
Obtain amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）The Poly(D,L-lactide-co-glycolide activated carboxylic thing 90-100 of preparation
Mg,
Step（2）The amination hyaluronic acid 130-150 mg of preparation, ultrasonic dissolution are added in 8-12 ml dichloromethane
5.9 ul DIPEAs, cross 40 μm of filter screen in 40-50 DEG C of magnetic agitation 12-48 h, product under nitrogen protection
The unreacted ammonification hyaluronic acid in part is removed, the product after filtration is proceeded to into bag filter, dialyse 72 h, obtains block copolymer；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）The block copolymer 3.0-5.0 mg of preparation, ultrasound are dissolved in 1-3
The DMSO and DMF of ml is according to volume ratio 3：In the mixed liquor of 1 mixing, at 20-25 DEG C, 500-800 r/min magnetic agitation 1-4
H, dropwise the deionized water of Deca 2-4 ml, continues 15 min-60min of stirring, proceeds in bag filter, and dialyse 12 h, obtains targeting
Self-assembled nanometer grain；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By sound sensitiser 1-5 g and step（4）The targeting self assembly of preparation is received
After grain of rice 5-100 g are dissolved in 3-5 ml acetone, 15-17 ml deionized waters, at 20-25 DEG C, 500-600 r/ are added dropwise over
2 ~ 4 h of min magnetic agitation, 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtain final product kernel load sound sensitiser
Nanoparticle；
Described sound sensitiser is arteannuin, dihydroarteannuin, artesunate, any one in arteether；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：Taking the chemotherapeutics of 6-12 mg, to be dissolved in 1-3 ml ultrapure
Water, in 22-25 DEG C, is dropwise added dropwise to step under 400-600 r/min stirrings（5）The kernel of preparation loads the nanoparticle of sound sensitiser
Middle lucifuge reacts 4-8 h, obtains the hud typed drug-carrying nanometer particle of cancer target self assembly；
Described chemotherapeutics are 5-ALA, 5-ALA methyl ester hydrochloride, levulic acid, acrylic acid
Methyl ester, 5- phthaloyl imino levulic acids, uroporphyrin prestox ester or coproporphyrin-One kind in trimethyl.
The application of the hud typed drug-carrying nanometer particle of cancer target self assembly prepared by methods described in antitumor drug is prepared.
Preparation process is simple of the present invention, easy to operate, abundant raw material source, low cost, toxic and side effects are little, it is easy to accomplish industry
Metaplasia is produced, and the hud typed drug-carrying nanometer particle of preparation-obtained cancer target self assembly carries arteannuin as drug-loading system, kernel, outward
Shell carries 5-ALA, it is possible to achieve the effective common load of arteannuin and 5-ALA.After nanoparticle enters cell, loaded change is discharged first
Medicine ALA is treated, synthesizes PpIX, Ultrasound-activated Hematoporphyrin Derivatives inducing apoptosis of tumour cell, so as to thin to pathological changes in cell mitochondrial
Born of the same parents play lethal effect.Then the arteannuin discharged by drug-loading system not only can produce carbon certainly under the mediation of heme
By base, and in the presence of extraneous ultrasound, substantial amounts of ROS is produced, and then is realized anti-swollen under 5-ALA and arteannuin federation policies
Tumor is acted on.The drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly has higher stability, physics and chemistry
The controllability of property；Realize that ligand binding promotes positioning and increase selectivity using surface functional group, promote medicine swollen
Tumor position Targeting delivery, so as to realize that sound is moved and chemotherapy combined antineoplastic action；Can the existing antitumor drug poison of effectively solving
Large side effects, poor biocompatibility, the problem of targeting difference.
Specific embodiment
With reference to embodiments the specific embodiment of the present invention is elaborated.
Embodiment 1
A kind of preparation method of the hud typed drug-carrying nanometer particle of cancer target self assembly, comprises the following steps：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.1 g end carboxyl poly (lactic acid-glycolic acid) copolymerization
Thing ultrasonic dissolution in 25 ml dichloromethane, add 40 mg N-hydroxy-succinamides and 75 mg 1- ethyls-（3- bis-
Dimethylaminopropyl）Carbodiimide hydrochloride, 12 h of magnetic agitation add the pre-cooling of 2 times of mixeding liquid volumes into mixed liquor
Absolute ether is precipitated, and is stored at room temperature 10 min, then is rinsed heavy with the isopyknic mixed liquor of absolute ether and absolute methanol of pre-cooling
Starch 3 times, is vacuum dried 72 h, obtains Poly(D,L-lactide-co-glycolide activated carboxylic thing；
（2）The preparation of amination hyaluronic acid：0.08 g hyaluronic acids ultrasound is weighed, the Methanamide of 15 ml is dissolved completely in
In, room temperature is cooled to, 500 mg EDC and 300 mg NHS is added, 8 h is stirred at room temperature, 10 ml ethylenediamines of Deca under ice bath
With Methanamide according to volume ratio 1：9 mixed liquor, after continuing ice bath 0.5 h of reaction, continues 1 h of stirring at room temperature into reaction
Liquid, adds the acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor, and crystallize, gained crystal must be filtered with 0.22 μm of organic membrane sucking filtration
Cake, then with washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h（MW：3500）, obtain amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）90 mg of Poly(D,L-lactide-co-glycolide activated carboxylic thing of preparation,
Step（2）130 mg of amination hyaluronic acid of preparation, ultrasonic dissolution add 5.9 ul in 10 ml dichloromethane
DIPEA, in 40 DEG C of 12 h of magnetic agitation under nitrogen protection, product is crossed 40 μm of filter screen and removes part unreacted
Ammonification hyaluronic acid, the product after filtration is proceeded to into bag filter（MW:12000-14000）, dialyse 72 h, obtains block copolymerization
Thing；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）3.0 mg of block copolymer of preparation, ultrasound are dissolved in 1 ml's
DMSO and DMF is according to volume ratio 3：In the mixed liquor of 1 mixing, at 20 DEG C, after 500 r/min magnetic agitation, 1 h, dropwise Deca 2
The deionized water of ml, continues 15 min of stirring, proceeds in bag filter（MW：8000-14000）, dialyse 12 h, obtains targeting self assembly
Nanoparticle；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By 1 g of sound sensitiser arteannuin and step（4）The targeting of preparation is from group
Accommodate after 20 g of the grain of rice is dissolved in 4 ml acetone, be added dropwise over 16 ml deionized waters, at 20 DEG C, 500 r/min magnetic agitation 2
H, 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtains final product the nanoparticle that kernel loads sound sensitiser；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：The chemotherapeutics 5-ALA for taking 6 mg is molten
In 1 ml ultra-pure waters, in 22 DEG C, under 400 r/min stirrings, step is dropwise added dropwise to（5）The kernel of preparation loads receiving for sound sensitiser
In the grain of rice, lucifuge reacts 4 h, obtains the hud typed drug-carrying nanometer particle of cancer target self assembly.
Embodiment 2
A kind of preparation method of the hud typed drug-carrying nanometer particle of cancer target self assembly, comprises the following steps：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.4 g polylactic acid acetate multipolymer ultrasounds molten
Solution in 30 ml dichloromethane, add 42 mg N-hydroxy-succinamides and 70 mg 1- ethyls-（3- dimethylaminos
Propyl group）Carbodiimide hydrochloride, 16 h of magnetic agitation add the absolute ether of the pre-cooling of 3 times of mixeding liquid volumes into mixed liquor
Precipitation, is stored at room temperature 10 min, then rinses precipitate 5 times with the isopyknic mixed liquor of absolute ether and absolute methanol of pre-cooling,
72 h are vacuum dried, Poly(D,L-lactide-co-glycolide activated carboxylic thing is obtained；
（2）The preparation of amination hyaluronic acid：0.15 g hyaluronic acids, 50 DEG C of oil baths are weighed, the formyl of 20 ml is dissolved completely in
In amine, room temperature is cooled to, adds 520 mg EDC and 315 mg NHS, 24 h are stirred at room temperature, 10 ml second two of Deca under ice bath
Amine is with Methanamide according to volume ratio 4：9 mixed liquor, after continuing ice bath reaction 2h, continues 4 h of stirring at room temperature into reactant liquor,
The acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor is added, crystallize, gained crystal obtain filter cake with 0.22 μm of organic membrane sucking filtration,
Again with washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h（MW：3500）, obtain amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）100 mg of Poly(D,L-lactide-co-glycolide activated carboxylic thing of preparation,
Step（2）150 mg of amination hyaluronic acid of preparation, ultrasonic dissolution add 5.9 ul in 8 ml dichloromethane
DIPEA, in 50 DEG C of 48 h of magnetic agitation under nitrogen protection, the filter screen removing part that product crosses 40 μm is not anti-
Product after filtration is proceeded to bag filter by the ammonification hyaluronic acid answered（MW:12000-14000）, dialyse 72 h, obtains block copolymerization
Thing；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）5.0 mg of block copolymer of preparation, ultrasound are dissolved in 3 ml's
DMSO and DMF is according to volume ratio 3：In the mixed liquor of 1 mixing, at 25 DEG C, after 800 r/min magnetic agitation, 4 h, dropwise drip
Plus 4 ml deionized water, continue stirring 60 min, proceed in bag filter（MW：8000-14000）, dialyse 12 h, obtains targeting certainly
Assemble nanometer grain；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By 5 g of sound sensitiser dihydroarteannuin and step（4）The targeting of preparation
After 25 g of self-assembled nanometer grain is dissolved in 5 ml acetone, 15 ml deionized waters are added dropwise over, at 25 DEG C, 600 r/min magnetic
Power stirs 4 h, and 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtains final product the nanometer that kernel loads sound sensitiser
Grain；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：Take the chemotherapeutics 5-ALA methyl ester of 12 mg
Hydrochlorate is dissolved in 3 ml ultra-pure waters, in 25 DEG C, is dropwise added dropwise to step under 600 r/min stirrings（5）The kernel load sound of preparation
In quick dose of nanoparticle, lucifuge reacts 8 h, obtains the hud typed drug-carrying nanometer particle of cancer target self assembly.
Embodiment 3
A kind of preparation method of the hud typed drug-carrying nanometer particle of cancer target self assembly, comprises the following steps：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.8g mono methoxy polyethylene glycol polylactic acid
Ethanol copolymer ultrasonic dissolution adds 50 mg N-hydroxy-succinamides and 80 mg 1- in 27 ml dichloromethane
Ethyl-（3- dimethylaminopropyls）Carbodiimide hydrochloride, 24 h of magnetic agitation add 3 times of mixing liquids into mixed liquor
The absolute ether precipitation of long-pending pre-cooling, is stored at room temperature 10 min, then isopyknic mixed with the absolute ether and absolute methanol of pre-cooling
Conjunction liquid flushing precipitate 5 times, is vacuum dried 72 h, obtains Poly(D,L-lactide-co-glycolide activated carboxylic thing；
（2）The preparation of amination hyaluronic acid：0.1 g hyaluronic acids ultrasound is weighed, is dissolved completely in the Methanamide of 17 ml,
Be cooled to room temperature, add 510 mg EDC and 310 mg NHS, 16 h are stirred at room temperature, under ice bath 10 ml ethylenediamines of Deca with
Methanamide is according to volume ratio 2：9 mixed liquor, after continuing ice bath 1 h of reaction, continues 3 h of stirring at room temperature into reactant liquor, then
The acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor, crystallize, gained crystal is added to obtain filter cake with 0.22 μm of organic membrane sucking filtration, then
With washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h（MW：3500）, obtain amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）95 mg of Poly(D,L-lactide-co-glycolide activated carboxylic thing of preparation,
Step（2）140 mg of amination hyaluronic acid of preparation, ultrasonic dissolution add 5.9 ul in 12 ml dichloromethane
DIPEA, in 45 DEG C of 24 h of magnetic agitation under nitrogen protection, the filter screen removing part that product crosses 40 μm is not anti-
Product after filtration is proceeded to bag filter by the ammonification hyaluronic acid answered（MW:12000-14000）, dialyse 72 h, obtains block copolymerization
Thing；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）4 mg of block copolymer of preparation, ultrasound are dissolved in 2 ml's
DMSO and DMF is according to volume ratio 3：In the mixed liquor of 1 mixing, at 22 DEG C, after 600 r/min magnetic agitation, 3 h, dropwise Deca 3
The deionized water of ml, continues 40 min of stirring, proceeds in bag filter（MW：8000-14000）, dialyse 12 h, obtains targeting self assembly
Nanoparticle；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By 2 g of sound sensitiser artesunate and step（4）The targeting of preparation is certainly
After 20 g of assemble nanometer grain is dissolved in 4 ml acetone, 15-17 ml deionized waters are added dropwise over, at 22 DEG C, 600 r/min magnetic
Power stirs 3 h, and 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtains final product the nanometer that kernel loads sound sensitiser
Grain；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：The chemotherapeutics levulic acid for taking 10 mg is dissolved in 2 ml
Ultra-pure water, in 24 DEG C, is dropwise added dropwise to step under 500 r/min stirrings（5）In the nanoparticle of the kernel load sound sensitiser of preparation
Lucifuge reacts 6 h, obtains the hud typed drug-carrying nanometer particle of cancer target self assembly.
The hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention is mainly used in the research of anti tumor activity in vitro, and
Jing tests achieve very satisfied Advantageous Effects, and relevant testing data is as follows：
Cancer target self assembly of the present invention hud typed drug-carrying nanometer particle is added in tumor cell and is cultivated, 4-6 h are administered
Ultrasound is focused on 2772AS type single screens comprehensive physiotherapy appearance afterwards（1 W/cm2, 40 S), it is subsequently placed into 37 DEG C, 5% CO2Incubator continues
20 h are incubated, the survival rate of tumor cell is determined afterwards.
Above-mentioned cancerous cell is:Pulmonary carcinoma, esophageal carcinoma, gastric cancer, hepatocarcinoma, breast carcinoma, ovarian cancer, cancer of pancreas, cervical cancer, thyroid
Cancer, intestinal tumor, carcinoma of gallbladder, renal carcinoma, carcinoma of prostate, carcinoma of penis, carcinoma of endometrium, choriocarcinoma, Hodgkin, non-Hodgkin′ses
Lymphoma, skin carcinoma, the one kind in malignant melanoma.
Correlation test data is as follows：
First, the measure of drug delivery system its drug loading of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention：
The drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention is taken, is surveyed by high performance liquid chromatography
The drug loading and envelop rate of fixed its sound sensitiser arteannuin and chemotherapeutics 5-ALA is respectively 0.928 mg/mL, 84 % and 1.78
mg/ml、75.71 %.Show that the drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention can conduct
The carrier of antitumor drug.
2nd, the particle size of the drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention and surface
The measure of carried charge：
The particle size and surface carried charge of the drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention
Measure, be measured using Nano-2590 type laser particle size analyzers, refractive index is set to 1.590, and absorptance is set to
0.010, temperature setting is 25 DEG C, and measurement pattern is set to automatically, using Z average statistical values as measurement result.Each level contracting
Fit to prepare 3 parts, per part of measurement once, takes the meansigma methodss of three measured values as measurement result.Dielectric constant is set to 79,
Coefficient of viscosity is set to 0.8872, and temperature setting is 25 DEG C, and measurement pattern is set to automatically.Each horizontal condensation body prepares 3
Part, per part of measurement once, takes the meansigma methodss of three measured values as measurement result.It is 100-300 that the result for measuring is particle diameter
Nm, current potential are 27.60 30 mV.
3rd, its extracorporeal anti-tumor of the drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention is lived
Property：
The anti tumor activity in vitro of the drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention, will
, as cancerous cell to be investigated with containing 10 % hyclones, 1 % is dual anti-for HePG2 hepatoma carcinoma cell (being provided by Shanghai cell bank)
DMEM culture medium culturings, are placed on 37 DEG C containing 5% CO2 Cell culture incubator in incubation, change liquid every other day, use 0.25 within 2-3 days
% trypsin contain 0.02 % EDTA) had digestive transfer culture once, carry out related experiment when cell length is to 70 %-80 %.All realities
The cell from exponential phase is tested, and it is carrier joint antineoplastic targeting self-assembled nanometer grain to be moved with srb assay detection sound
Toxic action of the drug delivery system to HePG2 cells.Take the logarithm trophophase cell, conventional digestion is to count after single cell suspension
Number, adjusts cell density to be measured to 5x103Individual/hole (edge hole is filled with aseptic PBS), spreads 96 orifice plates, and 200 ul are added per hole
Culture medium, is subsequently placed in 37 DEG C containing 5% CO2Cell incubation case in cultivate.After 24 h cells are all adherent, former training is discarded
Foster base, experimental group are separately added into Concentraton gradient for the hyaluronic acid targeted nano of (5,10,50,100 ug/mL) culture medium dilution
Grain carrier carries arteannuin and 5-ALA drug systems, and each concentration arranges 3-5 multiple holes, and blanc cell group adds blank cultures,
If 3-5 multiple holes, as a control group, continue to be placed in incubator and cultivate 24 h, terminate culture, gently add on culture fluid surface
50 % TCA of the pre-cooling of 25 ul are fixed, and after being stored at room temperature 5 min, then cell plates are moved to 4 DEG C of refrigerators placement l h, so may be used
Suspension cell is made to be fixed on the bottom of culture hole.Fixative is outwelled, each multiple holes is washed with deionized water 5 times, air-dried, it is completely dry
It is dry.The 0.4 % SRB dyeing liquors for adding 50 ul and being configured with 1 % acetic acid per hole, after room temperature lucifuge 20-30 min, outwell dyeing liquor,
Washed 5 times with 1 % acetic acid, remove unconjugated dyestuff.It is non-slow with 10 mmol/L (pH10.5), 150 ul/ holes after being air-dried
Rush TriS alkali dissolutions.After room temperature places 5 min, cell plates are placed and vibrates 5 min on the oscillator, it will improve the mixed of dyestuff
Conjunction property.Full-automatic microplate reader is can use to determine the absorbance of matched group and experimental group at 565 nm, 690 nm wavelength respectively.By public affairs
Formula：Cell inhibitory rate=1- (experimental group OD values/matched group OD values) 100 % of x calculate the cell inhibitory rate of each concentration.
Test result indicate that, the drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention can be led
Dynamic targets neoplastic cells or tissue, reduce its side effect to surrounding tissue, have given play to higher antitumor curative effect.
4th, using the drug delivery system of the hud typed drug-carrying nanometer particle of ultrasonic in combination cancer target self assembly to tumor cell
The measure of growth activity：
By the anti-tumor activity experiment in vitro of the drug delivery system of the hud typed drug-carrying nanometer particle of supersonic tumor targeting self assembly：
By HePG2 hepatoma carcinoma cell (being provided by Shanghai cell bank) as cancerous cell to be investigated, use dual anti-containing 10 % hyclones and 1 %
DMEM culture medium culturings, be placed on 37 DEG C containing 5% CO2Cell culture incubator in incubation, change liquid every other day, use within 2-3 days
0.25 % trypsin contain 0.02 % EDTA) had digestive transfer culture once, carry out related experiment when cell length is to 70-80 %.Institute
There is cell of the experiment from exponential phase.It is load with the dynamic joint antineoplastic targeting self-assembled nanometer grain of srb assay detection sound
Toxic action of the drug delivery system of body to HePG2 cells.Take the logarithm trophophase cell, after conventional digestion is single cell suspension
Count, cell density to be measured is adjusted to 5 x103Individual/hole (edge hole is filled with aseptic PBS), spreads 96 orifice plates, and 200 are added per hole
Ul culture medium, is subsequently placed in 37 DEG C containing 5% CO2Cell incubation case in cultivate.After 24 h cells are all adherent, original is discarded
Culture medium, experimental group be separately added into Concentraton gradient for (5,10,50,100 ug/mL) culture medium dilution with nanoparticle as carrier
The drug delivery system of hyaluronic acid targeting anti-tumor, each concentration arrange 3-5 multiple holes, and blanc cell group adds blank culture
Base, if 3-5 multiple holes are as a control group, continue to be placed in incubator and cultivates.4 h after ultrasound group dosing, with 2272AS type single screens
Comprehensive physiotherapy appearance ultrasound (1 W/cm2, 40 s), and ultrasound after terminating is placed in Tissue Culture Plate in incubator and continues 24 h of culture,
Terminate culture, the 50 % TCA of pre-cooling of 25 ul are gently added on culture fluid surface with fixation, after being stored at room temperature 5 min, then will
Cell plates move to 4 DEG C of refrigerators and place l h, can so make suspension cell be fixed on the bottom of culture hole.Fixative is outwelled, each is multiple
Hole is washed with deionized water 5 times, air-dries, is completely dried.The 0.4 % SRB dyeing liquors for adding 50 ul and being configured with 1 % acetic acid per hole, room
After warm lucifuge 20-30 min, dyeing liquor is outwelled, washed 5 times with 1 % acetic acid, remove unconjugated dyestuff.With 10 after being air-dried
150 ul/ holes non-buffered TriS alkali dissolutions of mmol/L (pH10.5).After room temperature places 5 min, cell plates are placed on into vibration
5 min are vibrated on device, it will improve the Combination of dyestuff.Full-automatic microplate reader is can use to distinguish at 565 nm, 690 nm wavelength
Determine the absorbance of matched group and experimental group.By formula：Carbazole alkaloid=1-(experimental group OD values/matched group OD values) X 100
% calculates the cell inhibitory rate of each concentration.
Test result indicate that, the drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly of the present invention can be loaded
Medicine enters inside tumor cells, has given play to the good efficacy of antitumor drug, and has combined 2272AS type single screen complex physiotherapies
After instrument ultrasound, can more obviously suppress tumor cell proliferation.
Preparation process is simple of the present invention, easy to operate, abundant raw material source, low cost, toxic and side effects are little, it is easy to accomplish industry
Metaplasia is produced, and the hud typed drug-carrying nanometer particle of preparation-obtained cancer target self assembly carries arteannuin as drug-loading system, kernel, outward
Shell carries 5-ALA, it is possible to achieve the effective common load of arteannuin and 5-ALA.After nanoparticle enters cell, loaded change is discharged first
Medicine ALA is treated, synthesizes PpIX, Ultrasound-activated Hematoporphyrin Derivatives inducing apoptosis of tumour cell, so as to thin to pathological changes in cell mitochondrial
Born of the same parents play lethal effect.Then the arteannuin discharged by drug-loading system not only can produce carbon certainly under the mediation of heme
By base, and in the presence of extraneous ultrasound, substantial amounts of ROS is produced, and then is realized anti-swollen under 5-ALA and arteannuin federation policies
Tumor is acted on.The drug delivery system of the hud typed drug-carrying nanometer particle of cancer target self assembly has higher stability, physics and chemistry
The controllability of property；Realize that ligand binding promotes positioning and increase selectivity using surface functional group, promote medicine swollen
Tumor position Targeting delivery, so as to realize that sound is moved and chemotherapy combined antineoplastic action；Can the existing antitumor drug poison of effectively solving
Large side effects, poor biocompatibility, the problem of targeting difference.
Claims (5)
1. the preparation method of the hud typed drug-carrying nanometer particle of a kind of cancer target self assembly, it is characterised in that comprise the following steps：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.1-0.8 g poly lactic-co-glycolic acids altogether
Polymers ultrasonic dissolution adds 42-50 mg N-hydroxy-succinamides and 70-80 mg 1- in 25-30 ml dichloromethane
Ethyl-（3- dimethylaminopropyls）Carbodiimide hydrochloride, magnetic agitation 12-24 h add 2-3 times and mix into mixed liquor
The absolute ether precipitation of the pre-cooling of liquid product, is stored at room temperature 10 min, then the absolute ether with pre-cooling and absolute methanol equal-volume
Mixed liquor rinse precipitate 3 ~ 5 times, be vacuum dried 72 h, obtain Poly(D,L-lactide-co-glycolide activated carboxylic thing；Described
Poly(D,L-lactide-co-glycolide is poly- for end carboxyl PLGA, polylactic acid acetate multipolymer or mono methoxy
One kind in ethylene glycol PLGA；
（2）The preparation of amination hyaluronic acid：0.08-0.15 g hyaluronic acids ultrasound or 50 DEG C of oil baths are weighed, is dissolved completely in
In the Methanamide of 15-20 ml, room temperature is cooled to, adds 500-520 mg EDC and 300-315 mg NHS, 8- is stirred at room temperature
24 h, under ice bath, Deca 10ml ethylenediamine and Methanamide are according to volume ratio 1 ~ 4：9 mixed liquor, continues ice bath reaction 0.5-2 h,
Continue stirring 1-4 h at room temperature into reactant liquor, add the acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor, crystallize, gained knot
0.22 μm of organic membrane sucking filtration of brilliant thing obtains filter cake, then with washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h, obtains
Amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）The Poly(D,L-lactide-co-glycolide activated carboxylic thing 90-100 of preparation
Mg,
Step（2）The amination hyaluronic acid 130-150 mg of preparation, ultrasonic dissolution are added in 8-12 ml dichloromethane
5.9 ul DIPEAs, cross 40 μm of filter screen in 40-50 DEG C of magnetic agitation 12-48 h, product under nitrogen protection
The unreacted ammonification hyaluronic acid in part is removed, the product after filtration is proceeded to into bag filter, dialyse 72 h, obtains block copolymer；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）The block copolymer 3.0-5.0 mg of preparation, ultrasound are dissolved in 1-3
The DMSO and DMF of ml is according to volume ratio 3：In the mixed liquor of 1 mixing, at 20-25 DEG C, 500-800 r/min magnetic agitation 1-4
H, dropwise the deionized water of Deca 2-4 ml, continues 15 min-60 min of stirring, proceeds in bag filter, and dialyse 12 h, obtains targeting
Self-assembled nanometer grain；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By sound sensitiser 1-5 g and step（4）The targeting self assembly of preparation is received
After grain of rice 5-100 g are dissolved in 3-5 ml acetone, 15-17 ml deionized waters, at 20-25 DEG C, 500-600 r/ are added dropwise over
2 ~ 4 h of min magnetic agitation, 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtain final product kernel load sound sensitiser
Nanoparticle；
Described sound sensitiser is arteannuin, dihydroarteannuin, artesunate, any one in arteether；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：Taking the chemotherapeutics of 6-12 mg, to be dissolved in 1-3 ml ultrapure
Water, in 22-25 DEG C, is dropwise added dropwise to step under 400-600 r/min stirrings（5）The kernel of preparation loads the nanometer of sound sensitiser
In grain, lucifuge reaction 4-8 h, obtain the hud typed drug-carrying nanometer particle of cancer target self assembly；
Described chemotherapeutics are 5-ALA, 5-ALA methyl ester hydrochloride, levulic acid, acrylic acid
Methyl ester, 5- phthaloyl imino levulic acids, uroporphyrin prestox ester or coproporphyrin-One kind in trimethyl.
2. the preparation method of the hud typed drug-carrying nanometer particle of a kind of cancer target self assembly according to claim 1, its feature
It is to comprise the following steps：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.1 g end carboxyl poly (lactic acid-glycolic acid) copolymerization
Thing ultrasonic dissolution in 25 ml dichloromethane, add 40 mg N-hydroxy-succinamides and 75 mg 1- ethyls-（3- bis-
Dimethylaminopropyl）Carbodiimide hydrochloride, 12 h of magnetic agitation add the pre-cooling of 2 times of mixeding liquid volumes into mixed liquor
Absolute ether is precipitated, and is stored at room temperature 10 min, then is rinsed heavy with the isopyknic mixed liquor of absolute ether and absolute methanol of pre-cooling
Starch 3 times, is vacuum dried 72 h, obtains Poly(D,L-lactide-co-glycolide activated carboxylic thing；
（2）The preparation of amination hyaluronic acid：0.08 g hyaluronic acids ultrasound is weighed, the Methanamide of 15 ml is dissolved completely in
In, room temperature is cooled to, 500 mg EDC and 300 mg NHS is added, 8 h is stirred at room temperature, 10 ml ethylenediamines of Deca under ice bath
With Methanamide according to volume ratio 1：9 mixed liquor, after continuing ice bath 0.5 h of reaction, continues 1 h of stirring at room temperature into reaction
Liquid, adds the acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor, and crystallize, gained crystal must be filtered with 0.22 μm of organic membrane sucking filtration
Cake, then with washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h, obtains amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）90 mg of Poly(D,L-lactide-co-glycolide activated carboxylic thing of preparation,
Step（2）130 mg of amination hyaluronic acid of preparation, ultrasonic dissolution add 5.9 ul in 10 ml dichloromethane
DIPEA, in 40 DEG C of 12 h of magnetic agitation under nitrogen protection, the filter screen removing part that product crosses 40 μm is not anti-
Product after filtration is proceeded to bag filter by the ammonification hyaluronic acid answered, and dialyse 72 h, obtains block copolymer；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）3.0 mg of block copolymer of preparation, ultrasound are dissolved in 1 ml's
DMSO and DMF is according to volume ratio 3：In the mixed liquor of 1 mixing, at 20 DEG C, after 500 r/min magnetic agitation, 1 h, dropwise Deca 2
The deionized water of ml, continues 15 min of stirring, proceeds in bag filter, and dialyse 12 h, obtains targeting self-assembled nanometer grain；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By 1 g of sound sensitiser arteannuin and step（4）The targeting of preparation is from group
Accommodate after 20 g of the grain of rice is dissolved in 4 ml acetone, be added dropwise over 16 ml deionized waters, at 20 DEG C, 500 r/min magnetic agitation 2
H, 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtains final product the nanoparticle that kernel loads sound sensitiser；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：The chemotherapeutics 5-ALA for taking 6 mg is molten
In 1 ml ultra-pure waters, in 22 DEG C, under 400 r/min stirrings, step is dropwise added dropwise to（5）The kernel of preparation loads receiving for sound sensitiser
In the grain of rice, lucifuge reacts 4 h, obtains the hud typed drug-carrying nanometer particle of cancer target self assembly.
3. the preparation method of the hud typed drug-carrying nanometer particle of a kind of cancer target self assembly according to claim 1, its feature
It is to comprise the following steps：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.4 g polylactic acid acetate multipolymer ultrasounds molten
Solution in 30 ml dichloromethane, add 42 mg N-hydroxy-succinamides and 70 mg 1- ethyls-（3- dimethylaminos
Propyl group）Carbodiimide hydrochloride, 16 h of magnetic agitation add the absolute ether of the pre-cooling of 3 times of mixeding liquid volumes into mixed liquor
Precipitation, is stored at room temperature 10 min, then rinses precipitate 5 times with the isopyknic mixed liquor of absolute ether and absolute methanol of pre-cooling,
72 h are vacuum dried, Poly(D,L-lactide-co-glycolide activated carboxylic thing is obtained；
（2）The preparation of amination hyaluronic acid：0.15 g hyaluronic acids, 50 DEG C of oil baths are weighed, the first of 20 ml is dissolved completely in
In amide, room temperature is cooled to, adds 520 mg EDC and 315 mg NHS, 24 h are stirred at room temperature, 10 ml second of Deca under ice bath
Diamidogen is with Methanamide according to volume ratio 4：9 mixed liquor, after continuing ice bath 2 h of reaction, continues 4 h of stirring at room temperature into reaction
Liquid, adds the acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor, and crystallize, gained crystal must be filtered with 0.22 μm of organic membrane sucking filtration
Cake, then with washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h, obtains amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）100 mg of Poly(D,L-lactide-co-glycolide activated carboxylic thing of preparation,
Step（2）150 mg of amination hyaluronic acid of preparation, ultrasonic dissolution add 5.9 ul in 8 ml dichloromethane
DIPEA, in 50 DEG C of 48 h of magnetic agitation under nitrogen protection, the filter screen removing part that product crosses 40 μm is not anti-
Product after filtration is proceeded to bag filter by the ammonification hyaluronic acid answered, and dialyse 72 h, obtains block copolymer；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）5.0 mg of block copolymer of preparation, ultrasound are dissolved in 3 ml's
DMSO and DMF is according to volume ratio 3：In the mixed liquor of 1 mixing, at 25 DEG C, after 800 r/min magnetic agitation, 4 h, dropwise drip
Plus 4 ml deionized water, continue stirring 60 min, proceed in bag filter, dialyse 12 h, obtain targeting self-assembled nanometer grain；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By 5 g of sound sensitiser dihydroarteannuin and step（4）The targeting of preparation
After 25 g of self-assembled nanometer grain is dissolved in 5 ml acetone, 15 ml deionized waters are added dropwise over, at 25 DEG C, 600 r/min magnetic
Power stirs 4 h, and 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtains final product the nanometer that kernel loads sound sensitiser
Grain；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：Take the chemotherapeutics 5-ALA methyl ester of 12 mg
Hydrochlorate is dissolved in 3 ml ultra-pure waters, in 25 DEG C, is dropwise added dropwise to step under 600 r/min stirrings（5）The kernel load of preparation
In the nanoparticle of sound sensitiser, lucifuge reacts 8 h, obtains the hud typed drug-carrying nanometer particle of cancer target self assembly.
4. the preparation method of the hud typed drug-carrying nanometer particle of a kind of cancer target self assembly according to claim 1, its feature
It is to comprise the following steps：
（1）The preparation of Poly(D,L-lactide-co-glycolide activated carboxylic thing：Weigh 0.8 g mono methoxy polyethylene glycol polylactic acid
Ethanol copolymer ultrasonic dissolution adds 50 mg N-hydroxy-succinamides and 80 mg 1- in 27 ml dichloromethane
Ethyl-（3- dimethylaminopropyls）Carbodiimide hydrochloride, 24 h of magnetic agitation add 3 times of mixing liquids into mixed liquor
The absolute ether precipitation of long-pending pre-cooling, is stored at room temperature 10 min, then isopyknic mixed with the absolute ether and absolute methanol of pre-cooling
Conjunction liquid flushing precipitate 5 times, is vacuum dried 72 h, obtains Poly(D,L-lactide-co-glycolide activated carboxylic thing；
（2）The preparation of amination hyaluronic acid：0.1 g hyaluronic acids ultrasound is weighed, is dissolved completely in the Methanamide of 17 ml,
Be cooled to room temperature, add 510 mg EDC and 310 mg NHS, 16 h are stirred at room temperature, under ice bath 10 ml ethylenediamines of Deca with
Methanamide is according to volume ratio 2：9 mixed liquor, after continuing ice bath 1 h of reaction, continues 3 h of stirring at room temperature into reactant liquor, then
The acetone cooling of the pre-cooling of 3 times of volumes of reactant liquor, crystallize, gained crystal is added to obtain filter cake with 0.22 μm of organic membrane sucking filtration, then
With washing with acetone filter cake 3 times, then redissolved with ultra-pure water, dialyse 48 h, obtains amination hyaluronic acid；
（3）The synthesis of block copolymer：Take step（1）95 mg of Poly(D,L-lactide-co-glycolide activated carboxylic thing of preparation,
Step（2）140 mg of amination hyaluronic acid of preparation, ultrasonic dissolution add 5.9 ul in 12 ml dichloromethane
DIPEA, in 45 DEG C of 24 h of magnetic agitation under nitrogen protection, the filter screen removing part that product crosses 40 μm is not anti-
Product after filtration is proceeded to bag filter by the ammonification hyaluronic acid answered, and dialyse 72 h, obtains block copolymer；
（4）The preparation of targeting self-assembled nanometer grain：Take step（3）4 mg of block copolymer of preparation, ultrasound are dissolved in 2 ml's
DMSO and DMF is according to volume ratio 3：In the mixed liquor of 1 mixing, at 22 DEG C, after 600 r/min magnetic agitation, 3 h, dropwise Deca 3
The deionized water of ml, continues 40 min of stirring, proceeds in bag filter, and dialyse 12 h, obtains targeting self-assembled nanometer grain；
（5）The preparation of the nanoparticle of kernel load sound sensitiser：By 2 g of sound sensitiser artesunate and step（4）The targeting of preparation is certainly
After 20 g of assemble nanometer grain is dissolved in 4 ml acetone, 15-17 ml deionized waters are added dropwise over, at 22 DEG C, 600 r/min magnetic
Power stirs 3 h, and 15000 r are centrifuged 30 min and must precipitate, and precipitation ultra-pure water redissolves, and obtains final product the nanometer that kernel loads sound sensitiser
Grain；
（6）The preparation of the hud typed drug-carrying nanometer particle of cancer target self assembly：The chemotherapeutics levulic acid for taking 10 mg is dissolved in 2 ml
Ultra-pure water, in 24 DEG C, is dropwise added dropwise to step under 500 r/min stirrings（5）In the nanoparticle of the kernel load sound sensitiser of preparation
Lucifuge reacts 6 h, obtains the hud typed drug-carrying nanometer particle of cancer target self assembly.
5. it is prepared by the hud typed drug-carrying nanometer particle of cancer target self assembly that prepared by claim 1 or any one of 2-4 methods described
Application in antitumor drug.
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| CN107115296A (en) * | 2017-07-05 | 2017-09-01 | 扬州大学 | A kind of preparation method of intermediate hydrophobic drug substance stable nano suspension |
| CN108635581A (en) * | 2018-05-23 | 2018-10-12 | 昆药集团股份有限公司 | A kind of composition and its application in the drug for preparing treatment tumour |
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| CN111956627A (en) * | 2020-09-25 | 2020-11-20 | 郑州大学 | Preparation method and application of drug compound with nano targeting effect for improving II type diabetes pancreatic microenvironment |
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| CN115554409A (en) * | 2022-10-12 | 2023-01-03 | 湖北科技学院 | High-molecular carrier material with mitochondrial targeting and preparation method and application thereof |
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| CN107095859A (en) * | 2017-04-24 | 2017-08-29 | 四川大学 | A kind of medicament-carried nano capsule sensitive with tumour cell bioreductive microenvironment and preparation method thereof |
| CN107095859B (en) * | 2017-04-24 | 2020-03-10 | 四川大学 | Drug-loaded nanocapsule with tumor cell bioreductive microenvironment sensitivity and preparation method thereof |
| CN107115296A (en) * | 2017-07-05 | 2017-09-01 | 扬州大学 | A kind of preparation method of intermediate hydrophobic drug substance stable nano suspension |
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| CN108635581A (en) * | 2018-05-23 | 2018-10-12 | 昆药集团股份有限公司 | A kind of composition and its application in the drug for preparing treatment tumour |
| CN109336993A (en) * | 2018-10-19 | 2019-02-15 | 苏州蔓尔生物科技有限公司 | A kind of 5-ALA hyaluronic acid ester and its preparation method and application |
| CN111956627A (en) * | 2020-09-25 | 2020-11-20 | 郑州大学 | Preparation method and application of drug compound with nano targeting effect for improving II type diabetes pancreatic microenvironment |
| CN111956627B (en) * | 2020-09-25 | 2022-02-18 | 郑州大学 | Preparation method and application of drug compound with nano targeting effect for improving II type diabetes pancreatic microenvironment |
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