CN104826127B - Light light and heat are dynamic to combine antineoplastic preparation method and application using folate-mediated gold nano star as the drug delivery system of carrier - Google Patents
Light light and heat are dynamic to combine antineoplastic preparation method and application using folate-mediated gold nano star as the drug delivery system of carrier Download PDFInfo
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- CN104826127B CN104826127B CN201510204171.0A CN201510204171A CN104826127B CN 104826127 B CN104826127 B CN 104826127B CN 201510204171 A CN201510204171 A CN 201510204171A CN 104826127 B CN104826127 B CN 104826127B
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Abstract
The present invention relates to the antineoplastic preparation method and application using folate-mediated gold nano star as the drug delivery system of carrier of the dynamic joint of light light and heat, existing antineoplastic toxic side effect can effectively be solved big, poor biocompatibility, the problem of targeting is poor, method is, gold nano star catalogue face is connected by covalent bond after amino and carboxyl with reactivity, then by acid amides reaction forming targeted molecular, obtain the gold nano star solution of active targeting；Chemically reacted again by the amino and near infrared fluorescent probe of target compound end, obtain the gold nano star solution with folate-targeted and near-infrared fluorescence imaging；Then antineoplastic adriamycin is directly loaded in gold nano star catalogue face by electrostatic adsorption again, produced, body physical and chemical stability of the present invention is good, preparation technology is simple and raw material sources are abundant, cost is low, and toxic side effect is small, can effectively suppress tumor cell proliferation.
Description
Technical field
The present invention relates to medicine, the dynamic joint of particularly a kind of smooth light and heat it is antineoplastic using folate-mediated gold nano star as
The preparation method and application of the drug delivery system of carrier.
Background technology
Photo-thermal therapy（Photo-thermal Therapy, PTT）It is the method by heating, changes residing for tumour cell
Environment, makes tumour cell denaturation, necrosis, reaches therapeutic purposes, for example, various gold nano structures, including gold nanorods,
Nanoshell, nanocages, the gold nano star of hollow nanosphere, especially branch-like, have been demonstrated there is surface near infrared region
Plasma resonance characteristic, thus hyperthermia is produced by near-infrared laser irradiation, so as to kill tumour cell.
Photodynamic therapy（Photodynamic Therapy, PDT）It is to absorb sensitising agent using tumor cells selectivity,
Then using the non-thermal laser irradiation diseased region of specific wavelength, the sensitising agent in tumor tissues is made to occur violent photochemistry anti-
Should, so that selective elemination tumour cell.It is a kind of noninvasive or minimal invasive, non-heat production, caused using photochemical reaction
Target tissue and the treatment method of target cell destruction.Gold nano star as a kind of excellent sensitising agent can near infrared light feelings
Active oxygen is produced under condition, peroxide, such as H is included2O2, singlet oxygen (Singlet Oxygen), epoxides
(epoxide) etc., and it is highly reactive that it produces active oxygen under light illumination, can show obvious cytotoxicity, than
Such as make the DNA break in cell, suppress cell mitogen and growth, the activity of cell protein hydrolase can also be suppressed.
Thus infer, the light light and heat that gold nano star can be used for deep tumor tissue move therapeutic alliance.
Gold nano star is because of its easily a large amount of synthesis and the big advantage of specific surface area, it is possible to increase contain medicament contg, meanwhile, by
There is height permeability to the near infrared light of 700-1100nm scopes in biosystem, and gold nano star has height within this range
The characteristic of absorption, therefore near-infrared laser thermotherapy can be carried out to tumour using its photothermal deformation characteristic within this range.It is logical
Cross the spaceborne antineoplastic formation Multifunctional centralized one of chemical modification gold nano i.e. chemotherapy, near-infrared thermotherapy and photodynamics connection
It is possible to close the drug delivery system for the treatment of.
Due to gold nano star in the case of high salt and some biomolecule (nucleic acid, protein etc.) are present easily occur aggregation and
Reunite, stability is poor.To improve the stability and biocompatibility of gold nano star, by the polyethylene glycol and polyetherimide of sulfhydrylation
The golden key of sulphur that amine is formed by sulfydryl and gold is connected to gold nano star catalogue face.By the water miscible poly- second two with long circulating characteristic
Alcohol is connected to gold nano star catalogue face, is not only able to utilization space steric effects, improves gold nano star stability, extends body-internal-circulation
Time, toxicity is reduced, and the different side-chain structures that polyethylene glycol is carried have provided for the targeting modification in gold nano star catalogue face
The decorating site of effect.In addition, sulfhydrylation PEI contains the nitrogen-atoms of the multistage ammonia of more protonation, can be in acidity
A large amount of trap protons in lysosome, cause the swelling of lysosome permeability, rupture, and then the medicine of endocytosis is promptly released into cell
Matter, realizes that lysosome is escaped.
Molecular targeted therapy is a kind of new oncotherapy means, can specifically act in tumor development and rise
The target molecule of key effect, or using the acceptor of tumour high-expression, by part by the targeted drug delivery of coupling to tumour
Cell, so as to reach treatment tumour, reduce the purpose of whole body toxic side effect.Folacin receptor is can be with mediated cell endocytosis, by leaf
A kind of high-affinity receptor of acid intake eukaryotic endochylema, enters clinical answer as a molecular target of oncotherapy
With research, its part folic acid has high affinity as targeting ligand to the folacin receptor that malignant cell excessive surface is expressed
Property, and hardly expressed in most normal structures, while by the pharmaceutical carrier of modification, be a kind of special targeting to
Medicine system, can active by drug delivery to specific position.
In tumour medicine research, using fluorescence probe labeled drug, directly medicine pair can be observed in live body level
Whether tumour targets, most preferably targeting time and medicine, in the accumulation of other organ-tissues of animal, is conducive to researcher most
The good time, carry out fabric analysis.The fluorescence probes such as indoles -5.5- cyanines-n-hydroxysuccinimide, indocyanine green, phthalocyanine are not
But it can be used in living imaging and the electronics near infrared light, molecule can be absorbed from ground state transition to sharp state, then excite
The molecule of state discharges heat, with thermotherapy effect.It is usually used in the fluorescence probe of living imaging, most of is traditional organic dyestuff
Molecule, such as indocyanine green, phthalocyanine, methylenum careuleum, rhodamine, fluorescein isothiocynate, and these organic dye molecules have it
Some shortcomings of, such as photostability itself is poor, and sensitivity is low and the defect such as targeting difference, thus limits it and further should
With.Drawbacks described above can be overcome by targeted nano granule carrier, the diagnosis and targeted therapy to tumour have a clear superiority.
Adriamycin is a kind of chemotherapeutics of wide spectrum, is clinically usually used in treating breast cancer, childhood solid tumor, soft tissue
Knurl, the malignant tumour such as nauseous lymph cancer.Clinical research in recent years finds that adriamycin has occurred being directed to tumour in clinical practice
The drug resistance of cell, the cytotoxicity simultaneously for normal structure limits its further application.If this kind of medicine is used for a long time
The most common toxicity of thing is injury of myocardium, can induce congestive heart failure.To overcome the above not enough, directly pass through Electrostatic Absorption
Effect（Au-N）The drug delivery system using gold nano star as carrier targeting anti-tumor can be built in gold nano star catalogue face by carrying adriamycin
System.
The drug delivery system using gold nano star as carrier targeting anti-tumor built at present, it is used as heat sensitizer and photosensitive
There is not been reported for application of the agent in tumor combined therapeutic, has the advantages that curative effect is fast, Small side effects, method are easy.Meanwhile, with
Gold nano star is that carrier load near infrared fluorescent dye can improve imaging time in the stability of fluorescent dye, extension body, and
It is assigned by carrier system to target.Application is wider compared with dyestuff direct imaging method, and application at present is concentrated mainly on
The living animal experimental stage in laboratory.Therefore, develop with the light light and heat on statistical significance and actual clinical meaning
It is dynamic to combine the technology that antineoplastic drug delivery system using folate-mediated gold nano star as carrier is desired solution in the industry
Problem.
The content of the invention
For above-mentioned situation, to overcome the defect of prior art, the purpose of the present invention is just to provide a kind of smooth light and heat and moved
Combine antineoplastic preparation method and application using folate-mediated gold nano star as the drug delivery system of carrier, can effectively solve
Certainly existing antineoplastic toxic side effect is big, poor biocompatibility, the problem of targeting is poor.
The technical scheme that the present invention is solved is that gold nano star catalogue face is connected into the ammonia with reactivity by covalent bond
After base and carboxyl, then by acid amides reaction forming targeted molecular, obtain the gold nano star solution of active targeting；Again by targetingization
The amino and near infrared fluorescent probe of compound end are chemically reacted, and are obtained with folate-targeted and near-infrared fluorescence imaging
Gold nano star solution；Then antineoplastic adriamycin is directly loaded in gold nano star catalogue face by electrostatic adsorption again, i.e.,
Obtaining folate-mediated multi-functional gold nano star is used for the dynamic joint anti-tumor drug transmission system of light light and heat, adriamycin and gold
The mass ratio of nanometer star is 1.5~3：1, specific preparation method is as follows：
（1）Prepare amidized gold nano star：Under the conditions of 18~25 DEG C, 300~720r/min magnetic agitation, add
Concentration is the 50 μ g/mL mL of gold nano star solution 10~20, then adds the amino chemical combination that concentration is 0.1~10.0 mg/mL
1~5mL of thing, carries out the h of lucifuge stirring reaction 6~12, then 10000 rpm high speed centrifugation 30min, is then redissolved with ultra-pure water
To amination gold nano star solution；
Described amino-compound is NH2One or two kinds of mixture in-PEG2000-SH and PEI-SH；
（2）Prepare the gold nano star with targeting：15~20mg of folic acid, N are weighed respectively, and N'- dicyclohexyls phosphinylidyne is sub-
10~15mg of amine, 7.5~10.5mg of n-hydroxysuccinimide, are then sequentially added in 2.5~5.0 mL reaction solutions, are mixed
Dissolving, then carry out activated carboxyl, i.e., lucifuge magnetic agitation reacts 8~24h under 40~50 DEG C, 300~720r/min, then will
The rpm high speed centrifugations 30min of resulting solution 10000 must be precipitated, and precipitation ultra-pure water redissolves, and add step（1）The amination of preparation
Acylation reaction, room temperature lucifuge 12~24h of stirring reaction, then 10000 rpm high speed centrifugation 30min are carried out in gold nano star solution,
Free small molecule is removed, the folate-targeted gold nano star solution for containing amino in end is obtained；
Described reaction solution is dimethyl sulfoxide (DMSO), phosphate buffer（It is neutral）With one kind in acetone；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：Take 0.01~2 mg fluorescence probes
In the reaction dissolvent for being dissolved in 0.01~1 mL, step is added（2）Acid amides is carried out in the folate-targeted gold nano star solution of preparation
Reaction, room temperature lucifuge 12~24h of stirring reaction, then 10000rpm ultracentrifugations 30min must be precipitated, and precipitation use ultra-pure water 0.01~
1mL redissolves, and obtains having targeting and the antineoplastic transmission system of near-infrared fluorescence imaging by carrier of gold nano star；
Described fluorescence probe is indoles -5- cyanines（It is abbreviated as cy5）, indoles -5.5- cyanines（It is abbreviated as cy5.5）With indoles-
5.5- cyanines-n-hydroxysuccinimide（It is abbreviated as cy5.5-NHS）In one kind；
Described reaction dissolvent is one kind in dimethyl sulfoxide (DMSO), phosphate buffer and acetone；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The target with near-infrared fluorescence imaging prepared
To gold nano star solution through dry powder, by powder and adriamycin with mass ratio 1:1.5~3.0 are sequentially added 10~40 mL
Reaction dissolvent in, under nitrogen protection, ultrasonic dissolution, at 18~25 DEG C 300-720r/min lucifuges stirring reaction 24~
48h, obtains mixed solution, then by obtained mixed solution with 10000rpm ultracentrifugations 30min（Remove unreacted adriamycin and
Dimethyl sulfoxide (DMSO)）, must precipitate, precipitation ultra-pure water redissolves, be drying to obtain the dynamic joint of light light and heat antineoplastic with folate-mediated
Gold nano star be carrier drug delivery system；
Described drying is one kind in being freeze-dried, be dried in vacuo and drying.
The light light and heat of above-mentioned preparation are dynamic to combine antineoplastic drug delivery using folate-mediated gold nano star as carrier
Application of the system in active targeting antineoplastic transport vehicle is prepared, or preparing antitumor smooth power or photo-thermal therapy
Application in heat sensitizer, or in the case where preparing near-infrared laser irradiation, triggering chemicals targets quick release in tumor tissues medicine
Application in thing, or prepare for light light and heat move joint anti-tumor drug transmission system preparation organism in living body fluorescent
It is imaged the application in medicine.
Folate-mediated multi-functional gold nano star of the present invention is used for light light and heat and moves joint anti-tumor drug biography
Delivery system, can load chemotherapeutics.Gold nano star has photothermal deformation characteristic and produces active oxygen ability, with reference to tumor locus
Laser irradiation is carried out to it, by the use of gold nano star as heat sensitizer, heat production can promote medicine in tumor locus under laser irradiation
Targeting delivery, so as to realize thermotherapy and chemotherapy combined antineoplastic action；Body physical and chemical stability of the present invention is good, prepares work
Skill is simple and raw material sources are abundant, and cost is low, and toxic side effect is small, can effectively suppress tumor cell proliferation, is that antineoplastic target is controlled
Innovation in treatment on pharmaceutical carrier.
Embodiment
The embodiment to the present invention elaborates with reference to embodiments.
The present invention can be provided in specific implementation by following examples.
Embodiment 1
The present invention can be realized in specific implementation by following steps：
（1）Prepare amidized gold nano star solution：Under the conditions of 25 DEG C, 540r/min magnetic agitation, concentration is added
For 50 μ g/mL gold nano star solution 20mL, the NH that concentration is 1mg/mL is then added2- PEG2000-SH solution 1mL, room temperature
Lucifuge stirring reaction 12h, then 10000rpm high speed centrifugation 30min, must be precipitated, and precipitation ultra-pure water 21mL redissolves, and obtains amination
Gold nano star（GNSTs-PEG）Solution；
（2）Prepare the gold nano star with targeting：Folic acid 19.91mg, N, N'- Dicyclohexylcarbodiimides are weighed respectively
13.94mg, n-hydroxysuccinimide 10.36mg（That is weight ratio：The hexamethylene base carbon acid imide ︰ N- hydroxyl ambers of leaf acid ︰ N, N'- bis-
The ︰ 0.52 of amber acid imide=1 ︰ 0.7）, then sequentially add in 4mL dimethyl sulfoxide (DMSO)s, mix dissolving, then carry out activated carboxyl, that is, exist
50 DEG C, lucifuge magnetic agitation reaction 12h under 480r/min, then must precipitate resulting solution 10000rpm high speed centrifugations 30min,
Precipitation ultra-pure water redissolves, and adds step（1）Acylation reaction is carried out in the amination gold nano star solution of preparation, room temperature lucifuge is stirred
Reaction 24h, then 10000 rpm high speed centrifugation 30min are mixed, free small molecule is removed, obtains the folic acid target for containing amino in end
To gold nano star（GNSTs-PEG-FA）Solution；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：Take 1mg fluorescence probes indoles-
5.5- cyanines-n-hydroxysuccinimide（cy5.5-HNS）It is dissolved in 1mL dimethyl sulfoxide (DMSO), adds step（2）The leaf of preparation
Acid amides reaction, room temperature lucifuge stirring reaction 24h, then 10000rpm ultracentrifugations 30min are carried out in acid targeting gold nano star solution
It must precipitate, precipitation ultra-pure water 0.5mL redissolves, be able to gold nano star has near-infrared fluorescence imaging and the gold of targeting for carrier
Nanometer star（GNS-PEG-FA-cy5.5）Solution；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）What is prepared has near-infrared fluorescence imaging and target
The gold nano star solution of tropism is freeze-dried to obtain powder, by powder and adriamycin with mass ratio 1:2 are sequentially added the two of 24mL
In methyl sulfoxide, under nitrogen protection, ultrasonic dissolution, 480r/min lucifuges stirring reaction 24h, obtains mixed solution at 25 DEG C,
Again by obtained mixed solution with 10000rpm ultracentrifugations 30min（Remove unreacted adriamycin and dimethyl sulfoxide (DMSO)）, obtain
Precipitation, precipitation ultra-pure water redissolves, and it is antineoplastic with folate-mediated gold nano to produce the dynamic joint of light light and heat with freeze-drying
Star is the drug delivery system of carrier（GNSTs/DOX-PEG-FA-cy5.5）Powder.
Embodiment 2
The present invention can be realized in specific implementation by following steps：
（1）Prepare amidized gold nano star：Under the conditions of 20 DEG C, 600r/min magnetic agitation, addition concentration is 50 μ
G/mL gold nano star solution 15mL, then adds the PEI-SH 3mL that concentration is 5mg/mL, carries out lucifuge stirring reaction 8h, then
10000 rpm high speed centrifugation 30min, are then redissolved with ultra-pure water and obtain amination gold nano star solution；
（2）Prepare the gold nano star with targeting：Folic acid 18mg, N, N'- Dicyclohexylcarbodiimides are weighed respectively
12mg, n-hydroxysuccinimide 9mg, are then sequentially added in 4mL phosphate buffers, mix dissolving, then carry out activation carboxylic
Base, i.e., lucifuge magnetic agitation reacts 16h under 45 DEG C, 500r/min, then by the rpm high speed centrifugations of resulting solution 10000
30min must be precipitated, and precipitation ultra-pure water redissolves, and add step（1）Carry out being acylated instead in the amination gold nano star solution of preparation
Should, room temperature lucifuge stirring reaction 16h, then 10000 rpm high speed centrifugation 30min, free small molecule is removed, end is obtained and contains
The folate-targeted gold nano star solution of amino；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：Take 1.5mg fluorescence probes indoles-
5- cyanines are dissolved in 0.8mL phosphate buffer, add step（2）Acyl is carried out in the folate-targeted gold nano star solution of preparation
Amine is reacted, and room temperature lucifuge stirring reaction 16h, then 10000rpm ultracentrifugations 30min must be precipitated, and precipitation is answered with ultra-pure water 0.8mL
It is molten, obtain that there is targeting and the antineoplastic transmission system of near-infrared fluorescence imaging by carrier of gold nano star；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The target with near-infrared fluorescence imaging prepared
Powder is obtained to gold nano star solution is vacuum dried, by powder and adriamycin with mass ratio 1:2.5 are sequentially added 35mL phosphoric acid
In salt buffer, under nitrogen protection, ultrasonic dissolution, 600r/min lucifuges stirring reaction 30h, obtains mixed solution at 20 DEG C,
Again by obtained mixed solution with 10000rpm ultracentrifugations 30min（Remove unreacted adriamycin and phosphate buffer）,
It must precipitate, precipitation ultra-pure water redissolves, it is antineoplastic with folate-mediated gold nano that vacuum drying produces the dynamic joint of light light and heat
Star is the drug delivery system powder of carrier.
Embodiment 3
The present invention can also be realized in specific implementation by following steps：
（1）Prepare amidized gold nano star：Under the conditions of 21 DEG C, 510r/min magnetic agitation, addition concentration is 50 μ
G/mL gold nano star solution 15mL, then adds the amino-compound 3mL that concentration is 5.0mg/mL, carries out lucifuge stirring reaction
9h, then 10000 rpm high speed centrifugation 30min, are then redissolved with ultra-pure water and obtain amination gold nano star solution；
（2）Prepare the gold nano star with targeting：Folic acid 17.5mg, N, N'- Dicyclohexylcarbodiimides are weighed respectively
12.5mg, n-hydroxysuccinimide 9mg, are then sequentially added in 3.5mL reaction solutions, mix dissolving, then carry out activation carboxylic
Base, i.e., lucifuge magnetic agitation reacts 16h under 45 DEG C, 510r/min, then by the rpm high speed centrifugations of resulting solution 10000
30min must be precipitated, and precipitation ultra-pure water redissolves, and add step（1）Carry out being acylated instead in the amination gold nano star solution of preparation
Should, room temperature lucifuge stirring reaction 18h, then 10000 rpm high speed centrifugation 30min, free small molecule is removed, end is obtained and contains
The folate-targeted gold nano star solution of amino；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：1mg fluorescence probes are taken to be dissolved in
In 0.5mL reaction dissolvent, step is added（2）Acid amides reaction is carried out in the folate-targeted gold nano star solution of preparation, room temperature is kept away
Light stirring reaction 18h, then 10000rpm ultracentrifugations 30min must be precipitated, and precipitation ultra-pure water 0.5mL redissolves, and obtains with Jenner
Meter Xing Wei carriers have targeting and the antineoplastic transmission system of near-infrared fluorescence imaging；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The target with near-infrared fluorescence imaging prepared
To gold nano star solution through dry powder, by powder and adriamycin with mass ratio 1:1.7 are sequentially added 25mL reaction dissolvent
In, under nitrogen protection, ultrasonic dissolution, the 510r/min lucifuges stirring reaction 36h at 21 DEG C obtains mixed solution, then will obtain
Mixed solution with 10000rpm ultracentrifugations 30min（Remove unreacted adriamycin and reaction dissolvent）, must precipitate, precipitation is used
Ultra-pure water redissolves, and is drying to obtain the antineoplastic drug delivery using folate-mediated gold nano star as carrier of the dynamic joint of light light and heat
System powder.
Embodiment 4
The present invention can also be realized in specific implementation by following steps：
（1）Prepare amidized gold nano star：Under the conditions of 20-22 DEG C, 500-600r/min magnetic agitation, add dense
The gold nano star solution 13-17mL for 50 μ g/mL is spent, the amino-compound 2-3mL that concentration is 6-8mg/mL is then added, carries out
Lucifuge stirring reaction 8-10 h, then 10000 rpm high speed centrifugation 30min, are then redissolved with ultra-pure water and obtain amination gold nano
Star solution；
（2）Prepare the gold nano star with targeting：Folic acid 17-18mg, N, N'- Dicyclohexylcarbodiimides are weighed respectively
12-13mg, n-hydroxysuccinimide 8-9.5mg, are then sequentially added in 3-4mL reaction solutions, mix dissolving, then lived
Change carboxyl, i.e., lucifuge magnetic agitation reacts 12-20h under 43-47 DEG C, 500-600r/min, then by resulting solution 10000
Rpm high speed centrifugations 30min must be precipitated, and precipitation ultra-pure water redissolves, and add step（1）In the amination gold nano star solution of preparation
Acylation reaction, room temperature lucifuge stirring reaction 16-20h, then 10000 rpm high speed centrifugation 30min are carried out, free small point is removed
Son, obtains the folate-targeted gold nano star solution for containing amino in end；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：Take 0.8-1.2mg fluorescence probes molten
Solution adds step in 0.4-0.6mL reaction dissolvent（2）Acid amides is carried out in the folate-targeted gold nano star solution of preparation anti-
Should, room temperature lucifuge stirring reaction 16-20h, then 10000rpm ultracentrifugations 30min must be precipitated, and precipitation uses ultra-pure water 0.4-0.6mL
Redissolve, obtain that there is targeting and the antineoplastic transmission system of near-infrared fluorescence imaging by carrier of gold nano star；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The target with near-infrared fluorescence imaging prepared
To gold nano star solution through dry powder, by powder and adriamycin with mass ratio 1:1.8-2.2 is sequentially added the anti-of 20-30mL
Answer in solvent, under nitrogen protection, ultrasonic dissolution, the 500-600r/min lucifuges stirring reaction 30-40h at 20-22 DEG C obtains mixed
Solution is closed, then by obtained mixed solution with 10000rpm ultracentrifugations 30min（Remove unreacted adriamycin and dimethyl is sub-
Sulfone）, must precipitate, precipitation ultra-pure water redissolves, be drying to obtain the dynamic joint of light light and heat antineoplastic with folate-mediated gold nano
Star is the drug delivery system powder of carrier.
The dynamic antineoplastic medicine using folate-mediated gold nano star as carrier of joint of light light and heat prepared by the present invention is passed
Delivery system particle diameter is 80-150nm, antitumor effective for active targeting as drug delivery carrier；As heat sensitizer anti-swollen
Knurl light power or photo-thermal therapy；Under near-infrared laser irradiation, triggering chemicals targeting quick release should in tumor tissues
With；Application in living body fluorescent imaging in vivo.And very satisfied advantageous effects are achieved through experiment, relevant examination
Certification of registered capital material is as follows：
The folate-mediated multi-functional gold nano star of the present invention is used for light light and heat and moves joint anti-tumor drug transmission system,
Application in antineoplaston is divided into vitro and in vivo two parts:
（1）In vitro:The folate-mediated multi-functional gold nano star of the present invention is used for the dynamic antineoplastic medicine of joint of light light and heat
Thing transmission system, which is added in tumour cell, is cultivated, the wide wavelength after administration after 4~6h with 700~1100nm wave-length coverages
Light source or 808nm laser lights are shone, 1~5min of light application time, are renewed fresh culture medium and are continued to be incubated 24h, determine tumour cell
Survival rate.
Above-mentioned cancer cell is:Organ surface or the internal various solid tumors occurred, lung cancer, nasopharyngeal carcinoma, cancer of the esophagus, stomach
Cancer, liver cancer, colorectal cancer, breast cancer, oophoroma, wing skin cancer, leukaemia, cancer of pancreas, cervical carcinoma, laryngocarcinoma, thyroid cancer, tongue cancer,
Brain tumor (intracranial tumors), intestinal tumor, gallbladder cancer, cholangiocarcinoma, kidney, prostate cancer, carcinoma of penis, emerald green ball tumour, endometrium
Cancer, choriocarcinoma, Primary Vaginal Carcinoma, Vulvar, Hodgkin's disease, NHL, cutaneum carcinoma, maligna
One kind in plain knurl.
（2）In vivo:The folate-mediated multi-functional gold nano star of the present invention is used for the dynamic antineoplastic medicine of joint of light light and heat
The intravenous injection of thing transmission system is arrived in tumor-bearing mice body, the wide wavelength light after administration after 4~6h with 700~1100nm wave-length coverages
Source or 808nm laser lights are shone, and 1~5min of light application time records the temperature of irradiated site with infrared thermal camera after irradiation.
Above-mentioned tumor-bearing mice is:Organ surface or the internal various solid tumors occurred, lung cancer, nasopharyngeal carcinoma, cancer of the esophagus, stomach
Cancer, liver cancer, colorectal cancer, breast cancer, oophoroma, wing skin cancer, leukaemia, cancer of pancreas, cervical carcinoma, laryngocarcinoma, thyroid cancer, tongue cancer,
Brain tumor (intracranial tumors), intestinal tumor, gallbladder cancer, cholangiocarcinoma, kidney, prostate cancer, carcinoma of penis, emerald green ball tumour, endometrium
Cancer, choriocarcinoma, Primary Vaginal Carcinoma, Vulvar, Hodgkin's disease, NHL, cutaneum carcinoma, maligna
One kind in plain knurl.
The new folate-mediated multi-functional gold nano star of the present invention is used for light light and heat and moves the transmission of joint anti-tumor drug
System can be made into multi-medicament formulation as heat sensitizer, such as injection, aseptic powder needle for injection, dispersant, patch, gel,
Implant etc..The preparation of the present invention can add various formulation additives, such as physiological saline, glucose, cushioning liquid and preservative
Deng.Administering mode can be intravenous injection, intramuscular injection, intratumor injection and hypodermic injection, cutaneous penetration, et al. Ke mode etc..
Correlation test data is as follows：
First, the folate-mediated multi-functional gold nano star of the present invention is used for the dynamic joint anti-tumor drug transmission system of light light and heat
Unite the measure of its drugloading rate：
Take the folate-mediated multi-functional gold nano star of the present invention to be used for light light and heat and move joint anti-tumor drug transmission system
System, by determined by ultraviolet spectrophotometry, its drugloading rate is 1.76mg/mL, shows the folate-mediated multi-functional Jenner of the present invention
Meter Xing be used for light light and heat move joint anti-tumor drug transmission system can as antineoplastic carrier.
2nd, the folate-mediated multi-functional gold nano star of the present invention is used for the dynamic joint anti-tumor drug transmission system of light light and heat
The particle size of system and the measure of surface carried charge：
Middle period of the invention acid mediated multi-functional gold nano star is used for light light and heat and moves the transmission of joint anti-tumor drug
The particle size of system and the measure of surface carried charge, are measured, refractive index is set using Nano-ZS90 type laser particle size analyzers
1.590 are set to, absorption coefficient is set to 0.010, and temperature setting is 25 DEG C, and measurement pattern is set to automatically, with Z average statistical values
It is used as measurement result.Each horizontal condensation body prepares 3 parts, and every part of measurement once, takes the average value of three measured values as survey
Measure result.Dielectric constant is set to 79, and coefficient of viscosity is set to 0.8872, and temperature setting is 25 DEG C, and measurement pattern is set to certainly
It is dynamic.Each horizontal condensation body prepares 3 parts, and every part of measurement once, takes the average value of three measured values as measurement result.Measure
Result be particle diameter be 80~150 nm, current potential is -20~30mV.
3rd, middle period of the invention acid mediated multi-functional gold nano star is used to light light and heat and moves joint anti-tumor drug transmitting
Its anti tumor activity in vitro of system：
Middle period of the invention acid mediated multi-functional gold nano star is used for light light and heat and moves the transmission of joint anti-tumor drug
The anti tumor activity in vitro of system, is used as cancer cell to be investigated by MCF-7 breast cancer cells (cell bank is provided by Shanghai).By people
Source breast cancer cell MCF-7, which is used, contains 10% hyclone, and 1% dual anti-RPMI1640 medium cultures are placed on 37 DEG C and contained
It is incubated in 5% CO2 cell culture incubator, changes liquid every other day, 3~4 days with 0.25% trypsase (containing 0.02% EDTA) had digestive transfer culture
Once, related experiment is carried out when cell length is to 70%~80%.The cell of exponential phase is selected in all experiments.Use srb assay
The folate-mediated multi-functional gold nano star of detection is used for light light and heat and moves joint anti-tumor drug transmission system to MCF-7 cells
Toxic action.Take the logarithm growth period cell, conventional digestion is to count after single cell suspension, adjust cell density to be measured to 5 ×
103Individual/hole (edge hole is filled with sterile PBS), spreads 96 orifice plates, and 200 μ l culture mediums are added per hole, 37 DEG C is subsequently placed in and contains
Cultivated in 5% CO2 cell incubation case.After 24 h cells are all adherent, former culture medium is discarded, experimental group is separately added into concentration
Gradient is（0.5,1,5,10,50,100 μ g/mL）The spaceborne Ti Zai adriamycins Department of Pharmacy of folate-targeted gold nano of culture medium dilution
System, each concentration sets 3~5 multiple holes, and blanc cell group adds blank cultures, if 3~5 multiple holes, as a control group, after
Continuous be placed in incubator cultivates 24 h, terminates culture, and 50% TCA that 25 μ l precooling is gently added on nutrient solution surface is fixed,
It is stored at room temperature after 5 min, then cell plates is moved into 4 DEG C of refrigerators and place 1 h, can so suspension cell is fixed on culture hole
Bottom.Fixer is outwelled, each multiple holes are washed with deionized water 5 times, air-dry, be completely dried.50 μ l are added to be configured with 1% acetic acid per hole
0.4% SRB dyeing liquors, after the min of room temperature lucifuge 20~30, outwell dyeing liquor, washed with 1% acetic acid 5 times, remove uncombined dye
Material.With the μ l/ holes non-buffered Tris alkali soluble solutions of 10 mmol/L (pH 10.5) 150 after being air-dried.Room temperature is placed after 5 min,
Cell plates are placed and vibrate 5 min on the oscillator, it will the Combination of dyestuff is improved.Can be with full-automatic ELIASA in 565
The absorbance of control group and experimental group is determined at nm, 690 nm wavelength respectively.By formula:Cell inhibitory rate=1- (experimental group OD
Value/control group OD values) × 100% calculate each concentration cell inhibitory rate.
Test result indicates that, it is antitumor that folate-mediated multi-functional gold nano star of the invention is used for the dynamic joint of light light and heat
Drug delivery system energy active targeting tumour cell or tissue, reduce its side effect to surrounding tissue, given play to higher
Antitumor curative effect.
4th, irradiate the folate-mediated multi-functional gold nano star of the present invention using laser and moved for light light and heat and combine antitumor
Drug delivery system to the measure of tumor cell growth activity：
Folate-mediated multi-functional gold nano star is irradiated by near-infrared laser antineoplastic for the dynamic joint of light light and heat
Drug delivery system in vitro test by antitumor activity：MCF-7 breast cancer cells (cell bank is provided by Shanghai) are used as to need checking
The cancer cell examined.People source breast cancer cell MCF-7 is used and contains 10% hyclone and 1% dual anti-RPMI1640 medium cultures,
It is placed on 37 DEG C and contains 5%CO2Cell culture incubator in be incubated, liquid, 3 are changed every other day~(contain 0.02% with 0.25% trypsase within 4 days
EDTA) had digestive transfer culture once, treats cell length to 70%~Related experiment is carried out when 80%.All experiments are from exponential phase
Cell.Detect that folate-mediated multi-functional gold nano star is used for light light and heat and moves joint anti-tumor drug transmission system with srb assay
The toxic action united to MCF-7 cells.Take the logarithm growth period cell, conventional digestion is to count after single cell suspension, is adjusted to be measured
Cell density is to 5 × 103Individual/hole (edge hole is filled with sterile PBS), spreads 96 orifice plates, 200 μ l culture mediums is added per hole, then
It is placed in 37 DEG C and contains 5%CO2Cell incubation case in cultivate.After 24h cells are all adherent, former culture medium is discarded, component is tested
Not Jia Ru concentration gradient be（0.5,1,5,10,50,100 μ g/mL）Culture medium dilution using gold nano star as carrier folate-targeted
Anti-tumor drug transmission system, each concentration sets 3~5 multiple holes, blanc cell group adds blank cultures, if 3~5 multiple
Hole, as a control group, continues to be placed in incubator and cultivates.4h after laser group dosing, is irradiated with 808nm laser（2W/cm2、
2min）, Tissue Culture Plate is placed in incubator by illumination after terminating to be continued to cultivate 24h, terminates culture, on nutrient solution surface gently
The 50%TCA for adding 25 μ l precooling is fixed, and is stored at room temperature after 5min, then cell plates are moved into 4 DEG C of refrigerators placement 1h, so may be used
Suspension cell is set to be fixed on the bottom of culture hole.Fixer is outwelled, each multiple holes are washed with deionized water 5 times, air-dried, it is completely dry
It is dry.Add the 0.4% SRB dyeing liquors that 50 μ l are configured with 1% acetic acid, room temperature lucifuge 20 per hole~After 30min, dyeing liquor is outwelled, with 1%
Acetic acid is washed 5 times, removes uncombined dyestuff.With the μ l/ holes non-buffered Tris alkali of 10mmol/L (pH 10.5) 150 after being air-dried
Dissolving.Room temperature is placed after 5min, and cell plates are placed and vibrate 5min on the oscillator, it will improve the Combination of dyestuff.It can use
Full-automatic ELIASA determines the absorbance of control group and experimental group respectively at 565nm, 690nm wavelength.By formula:Carbazole alkaloid
Rate=1- (experimental group OD values/control group OD values) × 100% calculates the cell inhibitory rate of each concentration.
Test result indicates that, it is antineoplastic that the folate-mediated multi-functional gold nano star of the present invention is used for the dynamic joint of light light and heat
Drug delivery system can load medicine and enter inside tumor cells, give play to the good efficacy of antineoplastic, and combine 808
After nm laser, it can more obviously suppress tumor cell proliferation.
5th, middle period of the invention acid mediated multi-functional gold nano star is used to light light and heat and moves joint anti-tumor drug transmitting
The measure of the internal antitumor activity of system：
By MCF-7 cells according to 1 × 107Individual/only, subcutaneous vaccination is under armpit before BALB/c nude mices are right.Nude inoculation swells
After knurl 7d, wherein 36 gross tumor volume >=100mm are taken3Nude mice, is randomly divided into 6 groups, every group 6.Specifically it is grouped as follows:(1) it is right
According to group (physiological saline group);(2) physiological saline laser group;(3) targeted drug group;(4) targeted drug laser group;(5) it is non-targeted
Medicine group;(6) non-targeted medicine laser group.6 groups using tail vein administration by the way of, wherein laser group administration 4h after irradiate
Tumor locus, the light source 808nm near-infrared light sources used, power is 2W/cm2, the once irradiating time is 1 min.It is administered every other day
Once, per injection physiological saline or drug solution or 100 μ g/mL the μ l of formulation soln 200, are administered 3 times altogether.Entirely
Daily observation nude mice animation, every other day claims its body weight and using the major diameter of vernier caliper measurement nude mice knurl in experimentation
(A) with minor axis (B), by formula V=1/2（A×B2）Calculate gross tumor volume.
It is demonstrated experimentally that under near-infrared laser irradiation, the folate-mediated multi-functional gold nano star of the present invention is used for into photo-thermal
The administration of joint anti-tumor drug transmission system is moved with light, can substantially suppress the increase of nude mouse tumor volume.
6th, middle period of the invention acid mediated multi-functional gold nano star is used to light light and heat and moves joint anti-tumor drug transmitting
The internal living imaging situation of system：
It is used for light light and heat using folate-mediated multi-functional gold nano star and moves joint anti-tumor drug transmission system
（GNSTs/DOX-PEG/PEI-FA-cy5.5）Carry out mouse imaging:Mouse is used after chloral hydrate anesthesia, in the tail of mouse
It is injected intravenously the cy5.5-NHS concentration of 5.0 μ L/g body weight molten for (50 μ g/mL) GNSTs/DOX-PEG/PEI-FA-cy5.5
Liquid, near-infrared fluorescence imaging is carried out using small animal living body imaging system under the irradiation of 808 nm laser.Then it is quiet that tail is distinguished again
Arteries and veins injects cy5.5-NHS（5.0 μ L/g, 50 μ g/mL）And GNSTs/DOX-PEG/PEI-FA-cy5.5（5.0 μ L/g, 50 μ
g/mL）Solution, in different time points, observes the near-infrared fluorescence imaging situation of two groups of nude mices in different Mice Bodies.
Interpretation of result, injects the mouse of GNSTs/DOX-PEG/PEI-FA-cy5.5 solution, after 1 minute, mouse surface
Skin sends stronger fluorescence signal than injecting cy5.5-NHS control group, and cardiac component sends obvious fluorescence signal, said
Bright GNSTs/DOX-PEG/PEI-FA-cy5.5 solution easily enters blood vessel, and easily enters mouse heart.Inject GNSTs/
During DOX-PEG/PEI-FA-cy5.5 solution 5 minutes, body surface fluorescence signal weakens, liver's signal enhancing.As injection GNSTs/
After DOX-PEG/PEI-FA-cy5.5 solution 4.5 hours, hepatic region fluorescence signal disappears, only in cardia member-retaining portion signal,
Remaining GNSTs/DOX-PEG/PEI-FA-cy5.5 solution enters intestines, and the fluorescence signal of cy5.5-NHS group mouse all turns
Move on to intestines.Result above shows, after mouse internal injection GNSTs/DOX-PEG/PEI-FA-cy5.5 solution, is more easy to enter
Heart and blood vessel, and the blood vessel of tumor locus has been laid well compared with normal structure more horn of plenty for the in-vivo imaging for tumour
Basis, present huge application value.
The present invention has advantageous effects following prominent compared with prior art:
（1）Small molecule folic acid of the present invention selection with good biocompatibility, tumor-targeting is decorating molecule, with ammonia
Base is linking arm, realizes with a kind of simple economy and that easily the method for industrialized production is modified gold nano star, will not be right
The characteristic of gold nano star in itself is destroyed, and the toxicity to organism is very low, and physics and chemical stability are good, can be in water
Placed three months in 4 DEG C of conditions in solution, significant change does not occur for its particle diameter, and preparation condition is gentle, reaction is simple, yield
Height, average single sintering 5L.
（2）The folate-mediated multi-functional gold nano star that the present invention is provided is used for light light and heat and moves joint anti-tumor drug
Transmission system, as a kind of good heat sensitizer of antineoplaston, test shows either external or internal in laser photograph
In the case of penetrating, the folate-mediated multi-functional gold nano star that the present invention is provided is used for light light and heat and moves joint anti-tumor drug biography
Delivery system can suppress the advolution of tumour cell and tissue well, and its inhibiting rate is 70% ~ 80%（The concentration of gold
0.5 ~ 1mg/kg, the concentration of adriamycin is 5 ~ 10mg/kg）And to normal cell and tissue toxic side effect very little.
（3）The folate-mediated multi-functional gold nano star that the present invention is provided is used for light light and heat and moves joint anti-tumor drug
Transmission system, as a kind of good sensitising agent, can produce active oxygen, and can show bright in the case of near infrared light
Aobvious cytotoxicity, more outstanding thermotherapy, phototherapy and chemotherapy synergistic antitumor effect can also have been given play to reference to antineoplastic
Really.
4）The folate-mediated multi-functional gold nano star that the present invention is provided is used for light light and heat and moves joint anti-tumor drug
Transmission system, can extend near infrared fluorescent dye circulation time in vivo（Up to 24 ~ 48 h）And improve fluorescent stability.From
And extend the observation window time to detecting object, the information of observation is fully obtained, the potentiality of living imaging is made it have, is anti-
Innovation on tumour medicine, economic and social benefit is huge.
Claims (9)
1. a kind of smooth light and heat are dynamic to combine antineoplastic system using folate-mediated gold nano star as the drug delivery system of carrier
Preparation Method, it is characterised in that connect in gold nano star catalogue face by covalent bond after amino and carboxyl with reactivity, then
By acid amides reaction forming targeted molecular, the gold nano star solution of active targeting is obtained；Pass through the ammonia of target compound end again
Base and near infrared fluorescent probe are chemically reacted, and obtain the gold nano star with folate-targeted and near-infrared fluorescence imaging molten
Liquid；Then antineoplastic adriamycin is directly loaded in gold nano star catalogue face by electrostatic adsorption again, that is, obtains folic acid Jie
The multi-functional gold nano star led is used for the matter that light light and heat move joint anti-tumor drug transmission system, adriamycin and gold nano star
Amount is than being 1.5~3：1, specific preparation method is as follows：
（1）Prepare amidized gold nano star：Under the conditions of 18~25 DEG C, 300~720r/min magnetic agitation, concentration is added
For the 50 μ g/mL mL of gold nano star solution 10~20, the amino-compound 1 that then addition concentration is 0.1~10.0 mg/mL~
5mL, carries out the h of lucifuge stirring reaction 6~12, then 10000 rpm high speed centrifugation 30min, is then redissolved with ultra-pure water and obtain amino
Change gold nano star solution；
Described amino-compound is NH2One or two kinds of mixture in-PEG2000-SH and PEI-SH；
（2）Prepare the gold nano star with targeting：15~20mg of folic acid, N, N'- Dicyclohexylcarbodiimides 10 are weighed respectively
~15mg, 7.5~10.5mg of n-hydroxysuccinimide, are then sequentially added in 2.5~5.0 mL reaction solutions, are mixed molten
Solution, then carry out activated carboxyl, i.e., lucifuge magnetic agitation reacts 8~24h under 40~50 DEG C, 300~720r/min, then by institute
Obtaining the rpm high speed centrifugations 30min of solution 10000 must precipitate, and precipitation ultra-pure water redissolves, and add step（1）The amination gold of preparation
Acylation reaction, room temperature lucifuge 12~24h of stirring reaction, then 10000 rpm high speed centrifugation 30min are carried out in nanometer star solution, are gone
Except free small molecule, the folate-targeted gold nano star solution for containing amino in end is obtained；
Described reaction solution is one kind in dimethyl sulfoxide (DMSO), phosphate buffer and acetone；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：0.01~2 mg fluorescence probes are taken to dissolve
In 0.01~1 mL reaction dissolvent, step is added（2）Acid amides reaction is carried out in the folate-targeted gold nano star solution of preparation,
Room temperature lucifuge 12~24h of stirring reaction, then 10000rpm ultracentrifugations 30min must be precipitated, and precipitation is multiple with 0.01~1mL of ultra-pure water
It is molten, obtain that there is targeting and the antineoplastic transmission system of near-infrared fluorescence imaging by carrier of gold nano star；
Described fluorescence probe is one in indoles -5- cyanines, indoles -5.5- cyanines and indoles -5.5- cyanines-n-hydroxysuccinimide
Kind；
Described reaction dissolvent is one kind in dimethyl sulfoxide (DMSO), phosphate buffer and acetone；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The gold of the targeting with near-infrared fluorescence imaging prepared
Nanometer star solution is through dry powder, by powder and adriamycin with mass ratio 1:1.5~3.0 are sequentially added the anti-of 10~40 mL
Answer in solvent, under nitrogen protection, ultrasonic dissolution, 300~720r/min lucifuges, 24~48h of stirring reaction at 18~25 DEG C,
Mixed solution is obtained, then obtained mixed solution must be precipitated with 10000rpm ultracentrifugation 30min, precipitation ultra-pure water redissolves,
It is drying to obtain the antineoplastic drug delivery system using folate-mediated gold nano star as carrier of the dynamic joint of light light and heat；
Described drying is one kind in being freeze-dried, be dried in vacuo and drying.
2. smooth light and heat according to claim 1 are dynamic to combine antineoplastic medicine using folate-mediated gold nano star as carrier
The preparation method of thing transmission system, it is characterised in that realized by following steps：
（1）Prepare amidized gold nano star solution：Under the conditions of 25 DEG C, 540r/min magnetic agitation, addition concentration is 50 μ
G/mL gold nano star solution 20mL, then adds the NH that concentration is 1mg/mL2- PEG2000-SH solution 1mL, room temperature lucifuge is stirred
Reaction 12h, then 10000rpm high speed centrifugation 30min are mixed, must be precipitated, precipitation ultra-pure water 21mL redissolves, and obtains amidized Jenner
Rice star solution；
（2）Prepare the gold nano star with targeting：Folic acid 19.91mg, N, N'- Dicyclohexylcarbodiimides are weighed respectively
13.94mg, n-hydroxysuccinimide 10.36mg, are then sequentially added in 4mL dimethyl sulfoxide (DMSO)s, mix dissolving, then lived
Change carboxyl, i.e., lucifuge magnetic agitation reacts 12h under 50 DEG C, 480r/min, then by resulting solution 10000rpm high speed centrifugations
30min must be precipitated, and precipitation ultra-pure water redissolves, and add step（1）Carry out being acylated instead in the amination gold nano star solution of preparation
Should, room temperature lucifuge stirring reaction 24h, then 10000 rpm high speed centrifugation 30min, free small molecule is removed, end is obtained and contains
The folate-targeted gold nano star solution of amino；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：Take 1mg fluorescence probe indoles -5.5- cyanines -
N-hydroxysuccinimide is dissolved in 1mL dimethyl sulfoxide (DMSO), adds step（2）The folate-targeted gold nano star solution of preparation
Middle progress acid amides reaction, room temperature lucifuge stirring reaction 24h, then 10000rpm ultracentrifugations 30min must be precipitated, precipitation ultra-pure water
0.5mL redissolves, and be able to gold nano star has near-infrared fluorescence imaging and the gold nano star solution of targeting for carrier；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）What is prepared has near-infrared fluorescence imaging and targeting
Gold nano star solution it is freeze-dried powder, by powder and adriamycin with mass ratio 1:2 are sequentially added 24mL dimethyl
In sulfoxide, under nitrogen protection, ultrasonic dissolution, the 480r/min lucifuges stirring reaction 24h at 25 DEG C obtains mixed solution, then will
Obtained mixed solution must be precipitated with 10000rpm ultracentrifugation 30min, and precipitation ultra-pure water redissolves, and is produced with freeze-drying
Light light and heat are dynamic to combine antineoplastic drug delivery system powder using folate-mediated gold nano star as carrier.
3. smooth light and heat according to claim 1 are dynamic to combine antineoplastic medicine using folate-mediated gold nano star as carrier
The preparation method of thing transmission system, it is characterised in that realized by following steps：
（1）Prepare amidized gold nano star：Under the conditions of 20 DEG C, 600r/min magnetic agitation, addition concentration is 50 μ g/mL
Gold nano star solution 15mL, then add concentration be 5mg/mL PEI-SH 3mL, carry out lucifuge stirring reaction 8h, then
10000 rpm high speed centrifugation 30min, are then redissolved with ultra-pure water and obtain amination gold nano star solution；
（2）Prepare the gold nano star with targeting：Folic acid 18mg, N, N'- Dicyclohexylcarbodiimides 12mg, N- are weighed respectively
HOSu NHS 9mg, is then sequentially added in 4mL phosphate buffers, mixes dissolving, then carries out activated carboxyl, that is, is existed
45 DEG C, lucifuge magnetic agitation reaction 16h under 500r/min, then must sink the rpm high speed centrifugations 30min of resulting solution 10000
Form sediment, precipitation ultra-pure water redissolves, add step（1）Acylation reaction is carried out in the amination gold nano star solution of preparation, room temperature is kept away
Light stirring reaction 16h, then 10000 rpm high speed centrifugation 30min, remove free small molecule, obtain the leaf that amino is contained in end
Acid targeting gold nano star solution；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：Take 1.5mg fluorescence probe indoles -5- cyanines
It is dissolved in 0.8mL phosphate buffer, adds step（2）Acid amides is carried out in the folate-targeted gold nano star solution of preparation anti-
Should, room temperature lucifuge stirring reaction 16h, then 10000rpm ultracentrifugations 30min must be precipitated, and precipitation ultra-pure water 0.8mL redissolves, and obtains
To the antineoplastic transmission system by carrier of gold nano star with targeting and near-infrared fluorescence imaging；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The gold of the targeting with near-infrared fluorescence imaging prepared
Nanometer star solution is vacuum dried to obtain powder, by powder and adriamycin with mass ratio 1:2.5 phosphate for being sequentially added 35mL delay
In fliud flushing, under nitrogen protection, ultrasonic dissolution, the 600r/min lucifuges stirring reaction 30h at 20 DEG C obtains mixed solution, then will
Obtained mixed solution must be precipitated with 10000rpm ultracentrifugation 30min, and precipitation ultra-pure water redissolves, and vacuum drying produces light
Light and heat are dynamic to combine antineoplastic drug delivery system powder using folate-mediated gold nano star as carrier.
4. smooth light and heat according to claim 1 are dynamic to combine antineoplastic medicine using folate-mediated gold nano star as carrier
The preparation method of thing transmission system, it is characterised in that realized by following steps：
（1）Prepare amidized gold nano star：Under the conditions of 21 DEG C, 510r/min magnetic agitation, addition concentration is 50 μ g/mL
Gold nano star solution 15mL, then add concentration be 5.0mg/mL amino-compound 3mL, carry out lucifuge stirring reaction 9h,
10000 rpm high speed centrifugation 30min, are then redissolved with ultra-pure water and obtain amination gold nano star solution again；
（2）Prepare the gold nano star with targeting：Folic acid 17.5mg, N, N'- Dicyclohexylcarbodiimides are weighed respectively
12.5mg, n-hydroxysuccinimide 9mg, are then sequentially added in 3.5mL reaction solutions, mix dissolving, then carry out activation carboxylic
Base, i.e., lucifuge magnetic agitation reacts 16h under 45 DEG C, 510r/min, then by the rpm high speed centrifugations of resulting solution 10000
30min must be precipitated, and precipitation ultra-pure water redissolves, and add step（1）Carry out being acylated instead in the amination gold nano star solution of preparation
Should, room temperature lucifuge stirring reaction 18h, then 10000 rpm high speed centrifugation 30min, free small molecule is removed, end is obtained and contains
The folate-targeted gold nano star solution of amino；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：1mg fluorescence probes are taken to be dissolved in 0.5mL
Reaction dissolvent in, add step（2）Acid amides reaction, the stirring of room temperature lucifuge are carried out in the folate-targeted gold nano star solution of preparation
React 18h, then 10000rpm ultracentrifugations 30min must precipitate, precipitation ultra-pure water 0.5mL redissolves, obtain using gold nano star as
Carrier has targeting and the antineoplastic transmission system of near-infrared fluorescence imaging；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The gold of the targeting with near-infrared fluorescence imaging prepared
Nanometer star solution is through dry powder, by powder and adriamycin with mass ratio 1:1.7 are sequentially added in 25mL reaction dissolvent,
Under nitrogen protection, ultrasonic dissolution, the 510r/min lucifuges stirring reaction 36h at 21 DEG C obtains mixed solution, then by obtained mixing
Solution must be precipitated with 10000rpm ultracentrifugation 30min, and precipitation ultra-pure water redissolves, and be drying to obtain the dynamic joint of light light and heat anti-
The drug delivery system powder using folate-mediated gold nano star as carrier of tumour.
5. smooth light and heat according to claim 1 are dynamic to combine antineoplastic medicine using folate-mediated gold nano star as carrier
The preparation method of thing transmission system, it is characterised in that realized by following steps：
（1）Prepare amidized gold nano star：Under the conditions of 20-22 DEG C, 500-600r/min magnetic agitation, adding concentration is
50 μ g/mL gold nano star solution 13-17mL, then adds the amino-compound 2-3mL that concentration is 6-8mg/mL, carries out lucifuge
Stirring reaction 8-10 h, then 10000 rpm high speed centrifugation 30min, then obtain amination gold nano star molten with ultra-pure water redissolution
Liquid；
（2）Prepare the gold nano star with targeting：Folic acid 17-18mg, N, N'- Dicyclohexylcarbodiimides 12- are weighed respectively
13mg, n-hydroxysuccinimide 8-9.5mg, are then sequentially added in 3-4mL reaction solutions, mix dissolving, then activated
Carboxyl, i.e., lucifuge magnetic agitation reacts 12-20h under 43-47 DEG C, 500-600r/min, then by the rpm of resulting solution 10000
High speed centrifugation 30min must be precipitated, and precipitation ultra-pure water redissolves, and add step（1）Enter in the amination gold nano star solution of preparation
Row acylation reaction, room temperature lucifuge stirring reaction 16-20h, then 10000 rpm high speed centrifugation 30min, remove free small molecule,
Obtain the folate-targeted gold nano star solution for containing amino in end；
（3）Prepare the gold nano star solution with near-infrared fluorescence imaging and targeting：0.8-1.2mg fluorescence probes are taken to be dissolved in
In 0.4-0.6mL reaction dissolvent, step is added（2）Acid amides reaction, room are carried out in the folate-targeted gold nano star solution of preparation
Warm lucifuge stirring reaction 16-20h, then 10000rpm ultracentrifugations 30min must be precipitated, and precipitation ultra-pure water 0.4-0.6mL redissolves,
Obtain that there is targeting and the antineoplastic transmission system of near-infrared fluorescence imaging by carrier of gold nano star；
（4）Prepare the gold nano star solution of load adriamycin：By step（3）The gold of the targeting with near-infrared fluorescence imaging prepared
Nanometer star solution is through dry powder, by powder and adriamycin with mass ratio 1:1.8-2.2 the reaction for being sequentially added 20-30mL is molten
In agent, under nitrogen protection, ultrasonic dissolution, the 500-600r/min lucifuges stirring reaction 30-40h at 20-22 DEG C must be mixed molten
Liquid, then obtained mixed solution must be precipitated with 10000rpm ultracentrifugation 30min, precipitation ultra-pure water redissolves, and is drying to obtain
Light light and heat are dynamic to combine antineoplastic drug delivery system powder using folate-mediated gold nano star as carrier.
6. the dynamic joint of light light and heat prepared by any one of claim 1 or 2-5 methods described is antineoplastic with folate-mediated gold
Nanometer star is application of the drug delivery system of carrier in active targeting antineoplastic transport vehicle is prepared.
7. the dynamic joint of light light and heat prepared by any one of claim 1 or 2-5 methods described is antineoplastic with folate-mediated gold
Nanometer star is application of the drug delivery system of carrier in the heat sensitizer of antitumor smooth power or photo-thermal therapy is prepared.
8. the dynamic joint of light light and heat prepared by any one of claim 1 or 2-5 methods described is antineoplastic with folate-mediated gold
Nanometer star for carrier drug delivery system in the case where preparing near-infrared laser irradiation, triggering chemicals targeting quick release is in swollen
Application in tumor tissue medicine.
9. the dynamic joint of light light and heat prepared by any one of claim 1 or 2-5 methods described is antineoplastic with folate-mediated gold
Nanometer star is used for the dynamic joint anti-tumor drug transmission system of light light and heat in preparation for the drug delivery system of carrier and prepared
Application in organism in living body fluorescent imaging medicine.
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| CN106421821A (en) * | 2016-10-08 | 2017-02-22 | 上海师范大学 | Multifunctional Cu3BiS3-PEG-(Ce6-Gd<3+>)-FA nano composite material, and preparation method and application thereof |
| CN108210506B (en) * | 2017-12-22 | 2020-08-25 | 上海交通大学 | pH response and polypeptide targeting nano-drug delivery carrier and preparation and application thereof |
| CN110038128B (en) * | 2019-04-25 | 2021-10-29 | 南京邮电大学 | Au @ Se @ Pt-Ce6 nano composite probe and preparation method thereof |
| CN110269942A (en) * | 2019-07-18 | 2019-09-24 | 上海交通大学医学院附属第九人民医院 | The targeting drug delivery system and synthetic method of a kind of photo-thermal in conjunction with chemotherapy |
| CN113583178B (en) * | 2020-07-31 | 2023-05-05 | 四川大学华西医院 | Branched sugar-containing polymer-based nanoparticle, and preparation method and application thereof |
| CN111999504B (en) * | 2020-08-10 | 2021-09-28 | 江南大学 | Mucin 1 and sialylglycosyl dual fluorescence imaging method and application thereof |
| CN113368238B (en) * | 2021-06-07 | 2022-08-02 | 青岛科技大学 | h-BN/MoS capable of realizing targeted photothermal and chemical synergistic treatment 2 Nano probe and preparation method and application thereof |
| CN113398112A (en) * | 2021-06-17 | 2021-09-17 | 西南医科大学 | FA-mediated BBA/CM-beta-CD targeted drug delivery system, preparation method and application thereof |
| CN115040647A (en) * | 2022-05-31 | 2022-09-13 | 山东大学 | Composite nano material for photo-thermal conversion and application thereof in preparation of anti-tumor preparation |
| CN115192708B (en) * | 2022-07-07 | 2024-02-27 | 安徽医科大学 | Nanocomposite loaded with antitumor drug, nano drug-carrying system, preparation and application |
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