CN105193831B - A kind of preparation method and applications for the self-assembling multifunctional nano target system for loading indocyanine green - Google Patents
A kind of preparation method and applications for the self-assembling multifunctional nano target system for loading indocyanine green Download PDFInfo
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- CN105193831B CN105193831B CN201510581283.8A CN201510581283A CN105193831B CN 105193831 B CN105193831 B CN 105193831B CN 201510581283 A CN201510581283 A CN 201510581283A CN 105193831 B CN105193831 B CN 105193831B
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- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 title claims abstract description 126
- 229960004657 indocyanine green Drugs 0.000 title claims abstract description 123
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 238000011068 loading method Methods 0.000 title claims description 14
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 78
- 229960004171 hydroxychloroquine Drugs 0.000 claims abstract description 45
- XXSMGPRMXLTPCZ-UHFFFAOYSA-N hydroxychloroquine Chemical compound ClC1=CC=C2C(NC(C)CCCN(CCO)CC)=CC=NC2=C1 XXSMGPRMXLTPCZ-UHFFFAOYSA-N 0.000 claims abstract description 42
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims abstract description 39
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Abstract
The present invention relates to the preparation method and applications of the self-assembling multifunctional nano target system of load indocyanine green,It is poor for the targeting of oncotherapy that existing antitumor drug can effectively be solved,Toxic side effect is big,Half-life short,Dosage is big,The problems such as function is single,Adriamycin is used as linking arm by disulfide bond and is connected to form self-assembled nanometer grain with hydroxychloroquine,Then by indocyanine green physical absorption into this nanoparticle,The self-assembling multifunctional nano target system of load indocyanine green is formed with phosphatide polyethylene glycol folic acid package nanoparticle again,The disulfide bond is 3,3 ' dithiodipropionic acids or 2,2 ' two thiodiglycolic acids,The molecular weight of phosphatide polyethylene glycol folic acid is 1 ~ 4kDa,Synthesis technology of the present invention is simple and convenient,With good biocompatibility,High targeting,Reduction-sensitive,The advantages that hypotoxicity,It is the innovation on diagnosing tumor treatment pharmaceutical preparation.
Description
Technical field
The present invention relates to medicine, particularly a kind of preparation for the self-assembling multifunctional nano target system for loading indocyanine green
Method and its application.
Background technology
In recent years, ascendant trend is presented in the incidence of malignant tumour.It is shown according to related data, China's Malignant Tumors among Residents are dead
It dies rate and adds 82.3% than the mid-1970s.As socio-economic development and living standards of the people improve, dietary structure changes
And aging of population, urbanization, Chronic Non-Communicable Diseases, which has become, causes main causes of death.Wherein, it is pernicious swollen
Knurl is one of current global major causes of death, has become and seriously endangers human life and health, restricts social economy's hair
One major class disease of exhibition.
Adriamycin (Doxorubicin, DOX) is a kind of antitumor antibiotics, the synthesis of RNA and DNA is can inhibit, to RNA
Inhibitory action it is most strong, antitumor spectra is wider, has effect to kinds of tumors, as breast cancer, lung cancer, soft tissue neoplasm, osteosarcoma,
Carcinoma of urinary bladder etc. belongs to cell cycle nonspecific agent (CCNSA), has killing effect to the tumour cell of various growth cycles.It has listed at present
Many toxic reactions are generated due to lacking targeting when doxorubicin hydrochloride injection clinically uses, such as cardiac toxic inhibits
Hemopoietic function of bone marrow and gastrointestinal reaction etc..
Hydroxychloroquine (Hydroxychloroquine, HCQ) is a kind of 4-aminoquinoline derivatives class antimalarial, main to use
In the suppressive prophylaxis of malaria control clinical symptoms and malaria.But some researches show that hydroxychloroquine is molten by inhibiting tumour cell
Enzyme body function and inhibit autophagy, autophagy is cell stress descend to maintain the metabolic processes of itself homeostasis various, and many is ground
Study carefully and show that the autophagy of chemotherapy induction has tumour protective effect, thus inhibit autophagy by tolerance of the reversing tumor to chemotherapy, tool
There is antitumor action.Thus hydroxychloroquine can increase its toxic action to tumour cell with chemotherapeutics such as adriamycin.
Indocyanine green (Indocyanine Green, ICG) is a kind of nir dye, is used as near infrared fluorescent dye
In life science and biomedical research there is important researching value, be common fluorescent marker in drug research.Base
In its unique property, indocyanine green is unique optical diagnostics factor with near-infrared characteristic of U.S. FDA approval, simultaneously
And a kind of preferable light absorber is used for carrying out the photo-thermal therapy of laser mediation.The dyestuff of this near-infrared can be used as one kind
Preferable contrast medium integrates the difunctional targeted probes of optical imagery and photo-thermal therapy to develop.
But the delivery system up to the present, clinically applied have lack targeting, half-life short, dosage it is big,
The shortcomings that toxic side effect is big, function is single.Have that strong targeting, long circulating, dosage be small, toxic side effect because the invention is a kind of
Small, multi-functional delivery system is a technical problem to be solved urgently.
The content of the invention
For the above situation, to overcome the defect of the prior art, the purpose of the present invention is just to provide a kind of load indoles cyanines
The preparation method and applications of green self-assembling multifunctional nano target system can effectively solve existing antitumor drug for swelling
Knurl treatment targeting is poor, toxic side effect greatly, half-life short, the problems such as dosage is big, function is single.
The technical solution that the present invention solves is that adriamycin (DOX) is used as linking arm and hydroxychloroquine (HCQ) by disulfide bond
Self-assembled nanometer grain is connected to form, then by indocyanine green (ICG) physical absorption into this nanoparticle, then with phosphatide polyethylene glycol
Folic acid (DSPE-PEG-FA) package nanoparticle forms the self-assembling multifunctional nano target system of load indocyanine green, described
Disulfide bond is 3,3 '-dithiodipropionic acid or 2,2 '-two thiodiglycolic acids, phosphatide polyethylene glycol folic acid (DSPE-PEG-FA)
Molecular weight is 1~4kDa (i.e. 1000-4000 dalton), is comprised the concrete steps that：
(1) self-assembled nanometer grain (DOX-S-S-HCQ) is synthesized：
1. being taken as the 3 of linking arm, 3 '-dithiodipropionic acid or 2,2 '-two thiodiglycolic acid 10mg~1g and dichloro are sub-
1.0~10.0ml of sulfone is placed in flask, adds in 15 μ lN, dinethylformamide (DMF), 85 DEG C are stirred at reflux 4h, and color is by shallow
Yellow becomes crocus；Extra thionyl chloride is removed with Rotary Evaporators, adds 0.5~5.0mlN, N- dimethyl formyls
Amine (DMF), obtains solution of acid chloride；
2. 0.1~1.0mmol of hydroxychloroquine is taken to be dissolved in 0.5~5.0ml reaction dissolvents, 20~200 μ l triethylamines are added in,
It is added drop-wise under ice bath stirring in solution of acid chloride, 2~8h is reacted under 35~85 DEG C of oil baths, obtain the hydroxychloroquine solution of chloride；
3. 0.1~1.0mmol adriamycins (DOX) are dissolved in 1.0~5.0ml reaction dissolvents, 20~200 μ l, tri- second is added in
Amine is added dropwise in the hydroxychloroquine solution of chloride under ice bath stirring, and 3~12h is stirred under 35~85 DEG C of oil baths, obtains reactant,
Reactant is dialysed 2 days with the bag filter of molecular cut off 1000, and dialyzate is ultra-pure water, it is every 3 it is small when change a water, freezing is dry
It is dry, derived from assemble nanometer grain (DOX-S-S-HCQ)；
The reaction dissolvent is formamide or N,N-dimethylformamide or N,N-dimethylformamide and formamide
Mixed solvent or the mixture of other similar solvents and other similar solvents；
(2) preparation of self-assembled nanometer particle loading indocyanine green (ICG/DOX-S-S-HCQ)：
1. weighing 0.5~10.0mg self-assembled nanometers grain (DOX-S-S-HCQ) is dissolved in 0.5~5.0ml dimethyl sulfoxide (DMSO)s
(DMSO) or in ethyl alcohol；
It 2. weighing indocyanine green (ICG) 0.1~5.0mg, is dissolved in 10~100ml ultra-pure waters, into indocyanine green solution；
3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, Probe Ultrasonic Searching 5 under ice bath~
30min must contain the self-assembled nanometer grain of indocyanine green (ICG)；
(3) nanoparticle (ICG/DOX-S-S- of phosphatide polyethylene glycol folic acid (DSPE-PEG-FA) package load indocyanine green
HCQ/DSPE-PEG-FA preparation)：
It weighs phosphatide polyethylene glycol folic acid (DSPE-PEG-FA) 0.1~5.0mg and is dissolved in the ethanol water that concentration is 1~8wt%
In 2~10ml of solution, 45~85 DEG C are heated to being completely dissolved, into phosphatide polyethylene glycol folic acid solution；Then indoles will be contained
The self-assembled nanometer grain of cyanines green (ICG) is added in phosphatide polyethylene glycol folic acid solution, be vortexed 1~5min, at room temperature stir 3~
8h dialyses solution 6~12h with the bag filter of molecular cut off 3500, it is every 2 it is small when change a water to get load indocyanine green
Self-assembling multifunctional nano target system (ICG/DOX-S-S-HCQ/DSPE-PEG-FA).
The self-assembling multifunctional nano target system (ICG/DOX-S-S-HCQ/ of load indocyanine green prepared by the present invention
DSPE-PEG-FA the treatment to tumour) is can be effectively used for, realizes the application in antitumor drug is prepared.
Synthesis technology of the present invention is simple and convenient, has good biocompatibility, high targeting, reduction-sensitive, low toxicity
The advantages that property, be the innovation on diagnosing tumor treatment pharmaceutical preparation.
Specific embodiment
It elaborates with reference to embodiments to the specific embodiment of the present invention.
Embodiment 1
The present invention in specific implementation, can be realized by following steps：
(1) self-assembled nanometer grain (DOX-S-S-HCQ) is synthesized：
1. being taken as the 2 of linking arm, 2 '-two thiodiglycolic acid 54.6mg and thionyl chloride 1.8ml are placed in round-bottomed flask,
15 μ lN are added in, dinethylformamide (DMF), 85 DEG C are stirred at reflux 4h, and color becomes crocus from light yellow；It is steamed with rotation
It sends out instrument and removes extra thionyl chloride, add 1ml n,N-Dimethylformamide (DMF), obtain solution of acid chloride；
2. hydroxychloroquine 0.3mmol is taken to be dissolved in 0.9mlN, in dinethylformamide (DMF), 60 μ l triethylamines, ice are added in
It is added drop-wise under bath stirring in solution of acid chloride, reacts 8h under 45 DEG C of oil baths, obtain the hydroxychloroquine solution of chloride；
3. 0.3mmol adriamycins (DOX) are dissolved in 1.8mlN, in dinethylformamide (DMF), 60 μ l, tri- second is added in
Amine is added dropwise in the hydroxychloroquine solution of chloride under ice bath stirring, 8h is stirred under 55 DEG C of oil baths, obtains reactant, and reactant is used
The bag filter of molecular cut off 1000 is dialysed 2 days, and dialyzate is ultra-pure water, it is every 3 it is small when change a water, be freeze-dried, derived from group
Accommodate the grain of rice (DOX-S-S-HCQ)；
(2) preparation of self-assembled nanometer particle loading indocyanine green (ICG/DOX-S-S-HCQ)：
1. it weighs 4mg self-assembled nanometers grain (DOX-S-S-HCQ) to be dissolved in 0.6ml dimethyl sulfoxide (DMSO)s；
It 2. weighing indocyanine green 0.6mg, is dissolved in 12ml ultra-pure waters, into indocyanine green solution；
3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, Probe Ultrasonic Searching 20min under ice bath is obtained
Contain the self-assembled nanometer grain of indocyanine green (ICG)；
(3) nanoparticle (ICG/DOX-S-S- of phosphatide polyethylene glycol folic acid (DSPE-PEG-FA) package load indocyanine green
HCQ/DSPE-PEG-FA preparation)：
It weighs phosphatide polyethylene glycol folic acid 0.6mg to be dissolved in the ethanol water 3ml that concentration is 4wt%, is heated to 55 DEG C
To being completely dissolved, into phosphatide polyethylene glycol folic acid solution；Then the self-assembled nanometer grain for containing indocyanine green (ICG) is added to
In phosphatide polyethylene glycol folic acid solution, vortex 3min stirs 6h at room temperature, and solution is saturating with the bag filter of molecular cut off 3500
Analyse 6h, it is every 2 it is small when change a water to get load indocyanine green self-assembling multifunctional nano target system (ICG/DOX-S-S-
HCQ/DSPE-PEG-FA)。
Embodiment 2
The present invention in specific implementation, can also be realized by following steps：
(1) self-assembled nanometer grain (DOX-S-S-HCQ) is synthesized：
1. being taken as the 3 of linking arm, 3 '-dithiodipropionic acid 84mg and thionyl chloride 2.4ml are placed in flask, add in 15
μ l n,N-Dimethylformamide, 85 DEG C are stirred at reflux 4h, and color becomes crocus from light yellow；It is removed with Rotary Evaporators more
Remaining thionyl chloride adds 1.5ml n,N-Dimethylformamide (DMF), obtains solution of acid chloride；
2. hydroxychloroquine 0.4mmol is taken to be dissolved in 1.2ml n,N-Dimethylformamide (DMF), 80 μ l triethylamines are added in,
It is added drop-wise under ice bath stirring in solution of acid chloride, reacts 6h under 55 DEG C of oil baths, obtain the hydroxychloroquine solution of chloride；
3. 0.4mmol adriamycins (DOX) are dissolved in 2.4ml n,N-Dimethylformamide (DMF), 80 μ l, tri- second is added in
Amine is added dropwise in the hydroxychloroquine solution of chloride under ice bath stirring, 10h is stirred under 45 DEG C of oil baths, obtains reactant, and reactant is used
The bag filter of molecular cut off 1000 is dialysed 2 days, and dialyzate is ultra-pure water, it is every 3 it is small when change a water, be freeze-dried, derived from group
Accommodate the grain of rice (DOX-S-S-HCQ)；
(2) preparation of self-assembled nanometer particle loading indocyanine green (ICG/DOX-S-S-HCQ)：
1. it weighs 6mg self-assembled nanometers grain (DOX-S-S-HCQ) to be dissolved in 0.8ml dimethyl sulfoxide (DMSO)s；
It 2. weighing indocyanine green (ICG) 0.8mg, is dissolved in 16ml ultra-pure waters, into indocyanine green solution；
3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, Probe Ultrasonic Searching 20min under ice bath is obtained
Contain the self-assembled nanometer grain of indocyanine green (ICG)；
(3) nanoparticle (ICG/DOX-S-S- of phosphatide polyethylene glycol folic acid (DSPE-PEG-FA) package load indocyanine green
HCQ/DSPE-PEG-FA preparation)：
It weighs phosphatide polyethylene glycol folic acid (DSPE-PEG-FA) 0.9mg and is dissolved in the ethanol water 4ml that concentration is 5wt%
In, 65 DEG C are heated to being completely dissolved, into phosphatide polyethylene glycol folic acid solution；Then will contain indocyanine green (ICG) from group
It accommodates the grain of rice to be added drop-wise in phosphatide polyethylene glycol folic acid solution, vortex 4min stirs 5h at room temperature, by solution molecular cut off
3500 bag filter dialysis 8h, it is every 2 it is small when change a water to get load indocyanine green self-assembling multifunctional nano target system
(ICG/DOX-S-S-HCQ/DSPE-PEG-FA)。
Embodiment 3
The present invention in specific implementation, can also be realized by following steps：
(1) self-assembled nanometer grain (DOX-S-S-HCQ) is synthesized：
1. being taken as the 3 of linking arm, 3 '-dithiodipropionic acid 105mg and thionyl chloride 3ml are placed in flask, add in 15 μ
L dimethylformamides (DMF), 85 DEG C are stirred at reflux 4h, and color becomes crocus from light yellow；It is extra to be removed with Rotary Evaporators
Thionyl chloride, add 2ml dimethylformamides (DMF), obtain solution of acid chloride；
2. hydroxychloroquine 0.5mmol is taken to be dissolved in 1.5ml formamides, 100 μ l triethylamines are added in, are added drop-wise under ice bath stirring
In solution of acid chloride, 4h is reacted under 75 DEG C of oil baths, obtains the hydroxychloroquine solution of chloride；
3. 0.5mmol adriamycins (DOX) are dissolved in 3ml formamides, 100 μ l triethylamines are added in, are added dropwise under ice bath stirring
In the hydroxychloroquine solution for entering chloride, 12h is stirred under 35 DEG C of oil baths, obtains reactant, reactant molecular cut off 1000
Bag filter dialyse 2 days, dialyzate is ultra-pure water, it is every 3 it is small when change a water, be freeze-dried, derived from assemble nanometer grain (DOX-S-S-
HCQ)；
(2) preparation of self-assembled nanometer particle loading indocyanine green (ICG/DOX-S-S-HCQ)：
1. it weighs 8mg self-assembled nanometers grain (DOX-S-S-HCQ) to be dissolved in 1ml dimethyl sulfoxide (DMSO)s (DMSO)；
It 2. weighing indocyanine green (ICG) 1mg, is dissolved in 20ml ultra-pure waters, into indocyanine green solution；
3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, Probe Ultrasonic Searching 20min under ice bath is obtained
Contain the self-assembled nanometer grain of indocyanine green (ICG)；
(3) nanoparticle (ICG/DOX-S-S- of phosphatide polyethylene glycol folic acid (DSPE-PEG-FA) package load indocyanine green
HCQ/DSPE-PEG-FA preparation)：
It weighs phosphatide polyethylene glycol folic acid (DSPE-PEG-FA) 1.2mg and is dissolved in the ethanol water 5ml that concentration is 6wt%
In, 75 DEG C are heated to being completely dissolved, into phosphatide polyethylene glycol folic acid solution；Then will contain indocyanine green (ICG) from group
It accommodates the grain of rice to be added drop-wise in phosphatide polyethylene glycol folic acid solution, vortex 5min stirs 3h at room temperature, by solution molecular cut off
3500 bag filter dialysis 10h, it is every 2 it is small when change a water to get load indocyanine green self-assembling multifunctional nano target system
It unites (ICG/DOX-S-S-HCQ/DSPE-PEG-FA).
It can be seen from the above, the present invention selects 3,3 '-dithiodipropionic acid (or 2, the analogs such as 2 '-two thiodiglycolic acids) is
Linking arm connects the adriamycin (DOX) containing amino and the hydroxychloroquine (HCQ) containing hydroxyl；DOX-S-S-HCQ in water may be used
To be self-assembly of nanoparticle, grain size can form after being mixed with indocyanine green (ICG) between 100~200nm and contain ICG
Nanoparticle.Then this nanoparticle is contained in phosphatide polyethylene glycol folic acid (DSPE-PEG-FA, 1~4kDa)：Disulfide bond is
Response type chemical bond is reduced, it is rapid to be broken so that antitumor drug by the effect of the glutathione of high expression in tumour cell
Efficiently release；Hydroxychloroquine has antitumor cell autophagocytosis, can cooperate with the antitumor cytotoxicity of enhancing adriamycin；Indocyanine green
With fluorescence imaging and photo-thermal therapy effect；In addition, medical surfaces carry out phosphatide polyethylene glycol folic acid (DSPE-PEG-FA, 1
~4kDa) after modification, the half-life period of delivery system can be extended and improve its stability in blood circulation, change administration system
The biodistribution of system, and with targeting.The present invention is that one kind has both cancer target, fluorescence imaging, and thermotherapy is mutually tied with chemotherapy
It closes, the newtype drug system ICG/DOX-S-S-HCQ/DSPE-PEG-FA that Clinics and Practices are integrated.And it is achieved through experiment non-
Often satisfied advantageous effects, related testing data are as follows：
1st, the self-assembling multifunctional nano target system of load indocyanine green (receive by the self-assembling multifunctional of load indocyanine green
Rice targeted system be also known as ICG/DOX-S-S-HCQ/DSPE-PEG-FA nanometer formulations) in DOX assay：
The content of DOX is measured using uv analysis method, the drugloading rate of sample is calculated with formula (1), drugloading rate reaches
36.0%,
2nd, the grain size of ICG/DOX-S-S-HCQ/DSPE-PEG-FA nanometer formulations and current potential characterization：
After appropriate ICG/DOX-S-S-HCQ/DSPE-PEG-FA nanometer formulations is taken to be diluted with water to suitable concentration, Nano- is used
It is respectively 130.2nm and -23.2 ± 0.30mv that ZS90 type laser nano Particle Size Analyzers, which measure its grain size and current potential,.
3rd, ICG/DOX-S-S-HCQ/DSPE-PEG-FA nanometer formulations test the Proliferation Ability of MCF-7 cells：
The step of being passed on according to cell is by MCF-7 cells processing to single cell suspension, the inoculation 5 × 10 per hole after counting3It is a
Cell on 96 orifice plates, incubator culture (37 DEG C, 5%CO2) for 24 hours, dosing after former culture medium is discarded after cell attachment is complete,
ICG/DOX-S-S-HCQ/DSPE-PEG-FA groups, DOX-S-S-HCQ groups and DOX groups, added drug concentration is with adriamycin
(DOX) concentration is formulated in a series of gradients with the culture medium without serum, and the concentration gradient of DOX is followed successively by 0,0.039,
0.078,0.156,0.313,0.625,1.25,2.5,5,10 μ g/ml, ICG/DOX-S-S-HCQ/DSPE-PEG-FA groups are equipped with
808nm laser control groups, and irradiate 808nm laser 3min (power 2.0) for a period of time after dosing, continue to take after cultivating 48h
Go out culture plate, 50% trichloroacetic acid of 50 μ l precoolings, final concentration of 10%, standing 5min are added in per hole；It moves to quiet in 4 DEG C of refrigerators
1h is put, is taken out, is washed 5 times with ultrapure, in air at room temperature after drying completely, the SRB50 μ l of 1% peracetic acid formulation are added in every hole,
20min is dyed at room temperature, outwells dye liquor, is washed 5 times with 1% acetic acid, is removed the dyestuff being not associated in hole, is used after air at room temperature drying
150 μ l of the 10mmol/lTris lye dissolvings of pH10.5, shake 10min, in enzyme-linked immunosorbent assay instrument in air heats shaking table
The upper light absorption angle value for measuring each hole at 515nm.Inhibiting rate (%)=(1- experimental group A/ control group As) × 100% is calculated, by
This show that the half-inhibition concentration (IC50) of above-mentioned sample is followed successively by：ICG/DOX-S-S-HCQ/DSPE-PEG-FA thermotherapy groups,
The non-thermotherapy groups of ICG/DOX-S-S-HCQ/DSPE-PEG-FA, DOX-S-S-HCQ groups, DOX groups are respectively 0.5249,1.0132,
1.0182,1.613μg/ml。
4th, ICG/DOX-S-S-HCQ/DSPE-PEG-FA nanometer formulations absorb MCF-7 cells streaming and test：
Experiment is divided into DOX-S-S-HCQ/DSPE-PEG-FA groups, DOX-S-S-HCQ groups, it is thin to be additionally provided with blank for DOX groups
Born of the same parents' group.The step of MCF-7 cells in growth period are passed on according to cell of taking the logarithm is processed into single cell suspension, and six are spread after counting
Orifice plate is 3 × 10 per hole cell number5A/hole, and incubator (37 DEG C, 5%CO2) in culture for 24 hours, it is adherent completely after, discard culture
Base adds in the drug prepared with the culture medium without serum, sets 0.5h, 1h, 2h respectively, and the period of 4h, 6h carry out the later stage
Processing, liquid sucking correspond in EP pipes, are washed 2 times with PBS, then with the pancreatin digestion process 1min without EDTA of 0.5ml, added
Enter culture medium 1ml, suck corresponding EP pipes, after 1000rpm/10min centrifugations, supernatant discarding, addition 2mlPBS is blown in precipitation
It beats uniformly, after 1000rpm/10min centrifugations, supernatant discarding in precipitation, is transferred to after adding in 0.5mlPBS piping and druming uniformly
Ice chest is placed in 1.5mlEP pipes, machine testing in streaming is waited, measures time point 6h and the streaming intake of MCF-7 is followed successively by：
59.4%, 75.2%, 90.3%.
5th, the pharmacodynamics test of ICG/DOX-S-S-HCQ/DSPE-PEG-FA nanometer formulations：
Prepare 10 female mices and carry out S-180 sarcoma ascites cultures, ascites is extracted after two weeks, for mouse entity knurl
Plantation, when the volume of tumour reaches 100mm3More than when, tumor model is inoculated with successfully, tumor-bearing mice is randomly divided into eight groups, often
Group eight, grouping situation are as follows：(1) blank group；(2) blank-laser group；(3) DOX groups；(4) DOX- laser groups；(5)DOX-S-
S-HCQ groups；(6) DOX-S-S-HCQ laser groups；(7) ICG/DOX-S-S-HCQ/DSPE-PEG-FA groups；(8)ICG/DOX-S-S-
HCQ/DSPE-PEG-FA laser groups.Then the next day of carrying out administration, dosage press people mouse dose lonvestion as 4mg/kg, tail vein
Drug administration by injection.The survival state and the amount of weighing knurl volume (gross tumor volume V=A × B of observation mouse daily2/ 2), Ran Houtong
Cross opposite knurl volume R=V/V0Variation (V0For the size of the previous day knurl volume is administered) evaluation Antitumor Activity of Drugs.Record
Statistics indicate that being compared at the end of administration with before administration, each group knurl volume inhibiting rate is followed successively by 0.02%, 2.59%, 47.36%,
53.56%th, 70.23%, 74.62%, 74.03%, 86.45%, have and significantly inhibit the effect of gross tumor volume, available for preparing
Antitumor drug.
Experiment shows compared with prior art, the present invention with advantageous effect following prominent：
1st, the bioreductive glutathione the present invention is based on high concentration provides tumor tissues reproducibility microenvironment, selection
Linking arm connection antineoplastic adriamycin and hydroxychloroquine containing disulfide bond, antineoplastic can be realized using this trigger mechanism
Object quick, Targeting delivery drug and reduces the toxic side effect of antitumor drug in vivo；In addition, the anti-autophagy of hydroxychloroquine
Effect is cooperateed with the antitumor action of adriamycin, can significantly increase lethality of the adriamycin to tumor tissues；
2nd, the present invention integrates optical imagery and photo-thermal therapy, and good effect can improve 10-30%；
3rd, the carrier that the present invention is formed has good biocompatibility, long circulating, Drug-loading Pattern is various, tumor-targeting is good
Deng being very beneficial for the treatment of tumour, effective percentage can be increased to more than 80%, be the big innovation in tumor, warp
Ji and social benefit are huge.
Claims (5)
- A kind of 1. preparation method for the self-assembling multifunctional nano target system for loading indocyanine green, which is characterized in that adriamycin Linking arm is used as by disulfide bond and is connected to form self-assembled nanometer grain with hydroxychloroquine, indocyanine green physical absorption is then arrived this In nanoparticle, then the self-assembling multifunctional nano target for loading indocyanine green is formed with phosphatide polyethylene glycol folic acid package nanoparticle System, the disulfide bond are 3,3 '-dithiodipropionic acid or 2,2 '-two thiodiglycolic acids, point of phosphatide polyethylene glycol folic acid Son amount is 1 ~ 4kDa, is comprised the concrete steps that：(1) self-assembled nanometer grain is synthesized：1. it is taken as the 3 of linking arm, 3 '-dithiodipropionic acid or 2,2 '-two thiodiglycolic acid 10mg ~ 1g and thionyl chloride 1.0 ~ 10.0ml is placed in flask, adds in 15 μ l n,N-Dimethylformamide, 85 DEG C are stirred at reflux 4h, and color becomes tangerine from light yellow Yellow；Extra thionyl chloride is removed with Rotary Evaporators, 0.5 ~ 5.0ml n,N-Dimethylformamide is added, it is molten to obtain acyl chlorides Liquid；2. 0.1 ~ 1.0mmol of hydroxychloroquine is taken to be dissolved in 0.5 ~ 5.0ml reaction dissolvents, 20 ~ 200 μ l triethylamines are added in, ice bath stirs It mixes down and is added drop-wise in solution of acid chloride, 2 ~ 8h is reacted under 35 ~ 85 DEG C of oil baths, obtain the hydroxychloroquine solution of chloride；3. 0.1 ~ 1.0 mmol adriamycins are dissolved in 1.0 ~ 5.0ml reaction dissolvents, 20 ~ 200 μ l triethylamines, ice bath stirring are added in Under be added dropwise in the hydroxychloroquine solution of chloride, 3 ~ 12 h are stirred under 35 ~ 85 DEG C of oil baths, obtain reactant, reactant retains The bag filter of molecular weight 1000 is dialysed 2 days, and dialyzate is ultra-pure water, it is every 3 it is small when change a water, be freeze-dried, received derived from assembling The grain of rice；The reaction dissolvent is mixed for formamide or N,N-dimethylformamide or N,N-dimethylformamide and formamide Bonding solvent；(2) preparation of self-assembled nanometer particle loading indocyanine green：1. it weighs 0.5 ~ 10.0mg self-assembled nanometer grains to be dissolved in 0.5 ~ 5.0ml dimethyl sulfoxide (DMSO)s or ethyl alcohol；It 2. weighing 0.1 ~ 5.0mg of indocyanine green, is dissolved in 10 ~ 100ml ultra-pure waters, into indocyanine green solution；3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, 5 ~ 30min of Probe Ultrasonic Searching under ice bath must be wrapped Carry the self-assembled nanometer grain of indocyanine green；(3) preparation of the nanoparticle of phosphatide polyethylene glycol folic acid package load indocyanine green：It weighs 0.1 ~ 5.0mg of phosphatide polyethylene glycol folic acid to be dissolved in 2 ~ 10ml of ethanol water that concentration is 1 ~ 8wt%, be heated to 45 ~ 85 DEG C to being completely dissolved, into phosphatide polyethylene glycol folic acid solution；Then the self-assembled nanometer grain for containing indocyanine green is added Into phosphatide polyethylene glycol folic acid solution, be vortexed 1 ~ 5min, stirs 3 ~ 8h at room temperature, and solution is saturating with molecular cut off 3500 Analyse bag dialyse 6 ~ 12h, it is every 2 it is small when change a water to get load indocyanine green self-assembling multifunctional nano target system.
- 2. the preparation method of the self-assembling multifunctional nano target system of load indocyanine green according to claim 1, It is characterized in that, is realized by following steps：(1) self-assembled nanometer grain is synthesized：1. being taken as the 2 of linking arm, 2 '-two thiodiglycolic acid 54.6mg and thionyl chloride 1.8ml are placed in round-bottomed flask, are added in 15 μ l n,N-Dimethylformamide, 85 DEG C are stirred at reflux 4h, and color becomes crocus from light yellow；It is removed with Rotary Evaporators Extra thionyl chloride adds 1ml n,N-Dimethylformamide, obtains solution of acid chloride；2. hydroxychloroquine 0.3mmol is taken to be dissolved in 0.9ml n,N-Dimethylformamide, 60 μ l triethylamines are added in, under ice bath stirring It is added drop-wise in solution of acid chloride, reacts 8h under 45 DEG C of oil baths, obtain the hydroxychloroquine solution of chloride；3. 0.3mmol adriamycins are dissolved in 1.8ml n,N-Dimethylformamide, 60 μ l triethylamines are added in, are dripped under ice bath stirring It adds in the hydroxychloroquine solution of chloride, stirs 8h under 55 DEG C of oil baths, obtain reactant, reactant molecular cut off 1000 Bag filter dialyse 2 days, dialyzate is ultra-pure water, it is every 3 it is small when change a water, be freeze-dried, derived from assemble nanometer grain；(2) preparation of self-assembled nanometer particle loading indocyanine green：1. it weighs 4mg self-assembled nanometer grains to be dissolved in 0.6ml dimethyl sulfoxide (DMSO)s；It 2. weighing indocyanine green 0.6mg, is dissolved in 12ml ultra-pure waters, into indocyanine green solution；3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, Probe Ultrasonic Searching 20min under ice bath must be contained The self-assembled nanometer grain of indocyanine green；(3) preparation of the nanoparticle of phosphatide polyethylene glycol folic acid package load indocyanine green：It weighs phosphatide polyethylene glycol folic acid 0.6mg to be dissolved in the ethanol water 3ml that concentration is 4wt%, is heated to 55 DEG C to complete Dissolving, into phosphatide polyethylene glycol folic acid solution；Then the self-assembled nanometer grain for containing indocyanine green is added to the poly- second two of phosphatide In alcohol folic acid solution, vortex 3min stirs 6h at room temperature, and by the bag filter dialysis 6h of solution molecular cut off 3500, every 2 is small When change a water to get load indocyanine green self-assembling multifunctional nano target system.
- 3. the preparation method of the self-assembling multifunctional nano target system of load indocyanine green according to claim 1, It is characterized in that, is realized by following steps：(1) self-assembled nanometer grain is synthesized：1. being taken as the 3 of linking arm, 3 '-dithiodipropionic acid 84mg and thionyl chloride 2.4ml are placed in flask, add in 15 μ l N,N-Dimethylformamide, 85 DEG C are stirred at reflux 4h, and color becomes crocus from light yellow；It is extra to be removed with Rotary Evaporators Thionyl chloride adds 1.5ml n,N-Dimethylformamide, obtains solution of acid chloride；2. hydroxychloroquine 0.4mmol is taken to be dissolved in 1.2ml n,N-Dimethylformamide, 80 μ l triethylamines are added in, under ice bath stirring It is added drop-wise in solution of acid chloride, reacts 6h under 55 DEG C of oil baths, obtain the hydroxychloroquine solution of chloride；3. 0.4mmol adriamycins are dissolved in 2.4ml n,N-Dimethylformamide, 80 μ l triethylamines are added in, are dripped under ice bath stirring It adds in the hydroxychloroquine solution of chloride, stirs 10h under 45 DEG C of oil baths, obtain reactant, reactant molecular cut off 1000 Bag filter dialyse 2 days, dialyzate is ultra-pure water, it is every 3 it is small when change a water, be freeze-dried, derived from assemble nanometer grain；(2) preparation of self-assembled nanometer particle loading indocyanine green：1. it weighs 6mg self-assembled nanometer grains to be dissolved in 0.8ml dimethyl sulfoxide (DMSO)s；It 2. weighing indocyanine green 0.8mg, is dissolved in 16ml ultra-pure waters, into indocyanine green solution；3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, Probe Ultrasonic Searching 20min under ice bath must be contained The self-assembled nanometer grain of indocyanine green；(3) preparation of the nanoparticle of phosphatide polyethylene glycol folic acid package load indocyanine green：It weighs phosphatide polyethylene glycol folic acid 0.9mg to be dissolved in the ethanol water 4ml that concentration is 5wt%, is heated to 65 DEG C to complete Dissolving, into phosphatide polyethylene glycol folic acid solution；Then the self-assembled nanometer grain for containing indocyanine green is added to the poly- second two of phosphatide In alcohol folic acid solution, vortex 4min stirs 5h at room temperature, and by the bag filter dialysis 8h of solution molecular cut off 3500, every 2 is small When change a water to get load indocyanine green self-assembling multifunctional nano target system.
- 4. the preparation method of the self-assembling multifunctional nano target system of load indocyanine green according to claim 1, It is characterized in that, is realized by following steps：(1) self-assembled nanometer grain is synthesized：1. being taken as the 3 of linking arm, 3 '-dithiodipropionic acid 105mg and thionyl chloride 3ml are placed in flask, add in 15 μ l bis- Methylformamide, 85 DEG C are stirred at reflux 4h, and color becomes crocus from light yellow；It is sub- that extra dichloro is removed with Rotary Evaporators Sulfone adds 2ml dimethylformamides, obtains solution of acid chloride；2. hydroxychloroquine 0.5mmol is taken to be dissolved in 1.5ml formamides, 100 μ l triethylamines are added in, acyl chlorides is added drop-wise under ice bath stirring In solution, 4h is reacted under 75 DEG C of oil baths, obtains the hydroxychloroquine solution of chloride；3. 0.5mmol adriamycins are dissolved in 3ml formamides, 100 μ l triethylamines are added in, chloride is added dropwise under ice bath stirring In hydroxychloroquine solution, 12 h are stirred under 35 DEG C of oil baths, obtain reactant, reactant is dialysed with the bag filter of molecular cut off 1000 2 days, dialyzate was ultra-pure water, it is every 3 it is small when change a water, be freeze-dried, derived from assemble nanometer grain；(2) preparation of self-assembled nanometer particle loading indocyanine green：1. it weighs 8mg self-assembled nanometer grains to be dissolved in 1ml dimethyl sulfoxide (DMSO)s；It 2. weighing indocyanine green 1mg, is dissolved in 20ml ultra-pure waters, into indocyanine green solution；3. self-assembled nanometer grain solution is added drop-wise in indocyanine green solution under stirring, Probe Ultrasonic Searching 20min under ice bath must be contained The self-assembled nanometer grain of indocyanine green；(3) preparation of the nanoparticle of phosphatide polyethylene glycol folic acid package load indocyanine green：It weighs phosphatide polyethylene glycol folic acid 1.2mg to be dissolved in the ethanol water 5ml that concentration is 6wt%, is heated to 75 DEG C to complete Dissolving, into phosphatide polyethylene glycol folic acid solution；Then the self-assembled nanometer grain for containing indocyanine green is added to the poly- second two of phosphatide In alcohol folic acid solution, vortex 5min stirs 3h at room temperature, and solution is dialysed 10h with the bag filter of molecular cut off 3500, and every 2 Hour changes self-assembling multifunctional nano target system of the water to get load indocyanine green.
- 5. the self-assembling multifunctional nano target system of load indocyanine green prepared by any one of claim 1 or 2-4 the method Application of the system in antitumor drug is prepared.
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| CN101428025B (en) * | 2008-10-24 | 2011-06-15 | 中国人民解放军第四军医大学 | New use of anti-impaludism medicament hydroxyl chloroquine |
| CN103690486B (en) * | 2013-12-27 | 2015-12-02 | 深圳先进技术研究院 | Indocyanine green nano-targeted liposome and preparation method and application thereof |
| CN104523723B (en) * | 2014-12-08 | 2017-09-12 | 中国人民解放军第四军医大学 | A kind of Mitochondrially targeted micella delivery system of reversible tumor drug resistance |
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