CN109010827A - Chlorambucil-poly-dopamine prodrug nanoparticle antitumor application thereof - Google Patents

Chlorambucil-poly-dopamine prodrug nanoparticle antitumor application thereof Download PDF

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CN109010827A
CN109010827A CN201810960221.1A CN201810960221A CN109010827A CN 109010827 A CN109010827 A CN 109010827A CN 201810960221 A CN201810960221 A CN 201810960221A CN 109010827 A CN109010827 A CN 109010827A
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chlorambucil
poly
nanoparticle
dopamine
prodrug nanoparticle
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杜畅
董常明
张蓉
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Shanghai Jiaotong University
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Shanghai Jiaotong University
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Abstract

The present invention relates to Chlorambucil-poly-dopamine prodrug nanoparticle antitumor application thereofs, specifically based on the mild photo-thermal treatment of Chlorambucil-poly-dopamine prodrug nanoparticle-seamless treatment technology of chemotherapy combined tumour.Compared with prior art, space-time manipulation may be implemented in nanoparticle of the invention in anti-tumor effect, reach pinpoint tumour ablation, and prodrug nanoparticle passive target may be implemented and be applied to tumor locus by enhancing infiltration and retention effect (EPR)；Tumor vascular expansion and cell membrane permeability can be enhanced by mild photo-thermal effect, be effectively facilitated the endocytosis of accumulation and infiltration and tumour cell of the nanoparticle in tumour；Photo-thermal treatment can further increase chemotherapy effect with inducing antitumor immunity, to generate synergistic antitumor effect in photo-thermal treatment-chemotherapy combined treatment.The present invention is that a new path is opened up in accurate treatment of cancer, has potential potential applicability in clinical practice.

Description

Chlorambucil-poly-dopamine prodrug nanoparticle antitumor application thereof
Technical field
The present invention relates to biomedicine technical fields, more particularly, to a kind of Chlorambucil-poly-dopamine prodrug nanometer The antitumor application thereof of particle.
Background technique
Cancer is seriously to jeopardize the major disease of human survival and social development.Although photo-thermal therapy and photo-thermal treatment-chemotherapy Synergistic treatment method has been realized in the complete ablation of solid tumor, but they are usually the near infrared light in high-intensitive and high dose It irradiates lower temperature and reaches 50 DEG C of implementation above, thus tumor locus often will appear serious scar and skin-burns, very To normal surrounding tissue damage phenomena such as.Worse, the remaining tumour cell of therapentic part tends not to eradicate completely, Lead oncogenic recurrence and transfer.These defects hinder photo-thermal therapy and its photo-thermal treatment-chemotherapy combined to treat normal direction clinical application, Realize the seamless ablation of tumour and no longer recur that still there is very big challenge.Therefore, by adjusting mild photo-thermal treatment and height The balance between chemotherapy is imitated, prepares a kind of near infrared absorbing polymeric prodrugs nanoparticle of high drug load, exploitation is based on before this The seamless treatment technology of the tumour of medicine nanoparticle, i.e., mild photo-thermal treatment-chemotherapy combined treatment technology have important clinical application Prospect provides effective approach in order to solve the above problem.
Literature search discovery, Zhuang Liu et al. people is in topic < 1D Coordination Polymer Nanofibers For Low-Temperature Photothermal Therapy > (the 1D coordination polymer nanometer for low temperature photo-thermal therapy Fiber) (Yu Yang, Wenjun Zhu, Ziliang Dong, Yu Chao, Lai Xu, * Meiwan Chen, * and in paper Zhuang Liu*, Adv.Mater.2017,29,1703588) report the coordination loaded jointly about gambogicacid and indoles cyanines The polymer nanofiber tumour that (light intensity 0.3W/cm2, light application time 20 minutes) induces under the irradiation of 808nm near-infrared laser The research of Apoptosis.Although serious scar does not occur in tumor locus, which cannot in photo-thermal therapy All solid tumors are melted completely, wherein only 2/5 tumour is eradicated completely in 14 days, are not able to satisfy wanting for clinical tumor radical cure It asks；And be confined to single photo-thermal and treat system, integrated synergisticing performance, which is treated, there is no chemotherapy and photo-thermal studies.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide one kind to be based on benzenebutanoic acid nitrogen Mustard-poly-dopamine prodrug nanoparticle antitumor application thereof, the side of the specifically mild seamless treatment tumour of photo-thermal treatment-chemotherapy combined Method, to solve to induce tumor locus temperature excessively high under high-intensitive and high dose near infrared light in original technology, tumour Position will appear serious scar, and the damage of skin-burns and normal surrounding tissue and the recurrence of tumour and transfer etc. are clinical Using problem.
The purpose of the present invention can be achieved through the following technical solutions:
Present invention firstly provides a kind of Chlorambucil-poly-dopamine prodrug nanoparticle, the nano particle structure formulas It is as follows:
Wherein, n, m, o are positive integer, and n, m, o are identical or not identical.
The present invention further provides the Chlorambucil with above structure-poly-dopamine prodrug nanoparticle synthesis sides Method: Chlorambucil-dopamine conjugate is synthesized first, is then with the Chlorambucil of synthesis-dopamine conjugate molecules Raw material under alkaline condition with dopamine hydrochloride in the in the mixed solvent of distilled water and trishydroxymethylaminomethane, reacts, Dialysis, obtains Chlorambucil-poly-dopamine prodrug nanoparticle.
Chlorambucil-dopamine conjugate synthetic method, comprising the following steps:
Step 1,3,4- dihydroxyphenyl propionic acids are reacted with phosphorus trichloride, prepare 2,2- dimethyl -1,3- benzo dioxa Cyclopentene -5- propionic acid；
Step 2,2,2- dimethyl -1,3- benzodioxole -5- propionic acid, dicyclohexylcarbodiimide, 4- diformazan Aminopyridine reaction adds the reaction of 2- hydroxyethyl disulfide, obtains 2- (2- hydroxyethyl) disulfide group) ethyl -2,2- bis- Methyl-1,3- benzodioxole -5- ethyl propionate；
Step 3, Chlorambucil, dicyclohexylcarbodiimide, 4-dimethylaminopyridine reaction, adds 2- (2- hydroxyl Ethyl) disulfide group) ethyl -2,2- dimethyl -1,3- benzodioxole -5- ethyl propionate, is reacted, obtains ((2- (2,2- dimethyl -1,3- benzodioxole -5- propiono) oxygroup) ethyl) -4- (4- (bis- (2- chloroethyl) amino) Phenyl) methyl butyrate；
Step 4, ((2- (2,2- dimethyl -1,3- benzodioxole -5- propiono) oxygroup) ethyl) -4- (4- (bis- (2- chloroethyl) amino) phenyl) methyl butyrate reacts with trifluoroacetic acid, obtain Chlorambucil-dopamine conjugate point Son.
In an embodiment of the invention, step 1 method particularly includes: the acetone of 3,4- dihydroxyphenyl propionic acids Phosphorus trichloride is slowly added dropwise in solution, and after reaction, rotary evaporation is extracted with the mixture of water and ether, takes organic layer, rotation Turn evaporation, obtains white solid 2,2- dimethyl -1,3- benzodioxole -5- propionic acid.
In an embodiment of the invention, when carrying out step 1, the quality (mg) one of 3,4- dihydroxyphenyl propionic acids As be 2-3 times, preferably 2.61 times of volume (μ L) of phosphorus trichloride.
In an embodiment of the invention, when carrying out step 1,3,4- dihydroxyphenyl propionic acids and phosphorus trichloride are anti- The condition answered are as follows: 0 DEG C is reacted 6 hours.
In an embodiment of the invention, when carrying out step 1, based on mg and mL unit, 3,4- dihydroxy phenyls third The quality selection of acid is that the volume of acetone is preferably 50 times.
In an embodiment of the invention, when carrying out step 1, in the mixture of water and ether, the body of water and ether Product does not limit specific ratio than being arbitrary proportion.
In an embodiment of the invention, step 2 method particularly includes: 2, the 2- dimethyl-that is obtained by step 1 1,3- benzodioxole -5- propionic acid, dicyclohexylcarbodiimide, the dichloromethane solution of 4-dimethylaminopyridine, instead The dichloromethane solution of 2- hydroxyethyl disulfide is slowly added dropwise in Ying Hou, is leaching with methylene chloride and methyl alcohol mixed liquor after reaction The purification of washing lotion column separation, rotary evaporation is dry, obtains light yellow solid 2- (2- hydroxyethyl) disulfide group) ethyl -2,2- bis- Methyl-1,3- benzodioxole -5- ethyl propionate.
In an embodiment of the invention, when carrying out step 2,2- hydroxyethyl disulfide, dicyclohexyl carbon two are sub- Amine, 4-dimethylaminopyridine mole dosage be respectively selected as 2,2- dimethyl -1,3- benzodioxole -5- propionic acid and rub 1.2 times of that dosage.
In an embodiment of the invention, when carrying out step 2,2,2- dimethyl -1,3- benzo dioxanes penta Alkene -5- propionic acid, dicyclohexylcarbodiimide, the dichloromethane solution of 4-dimethylaminopyridine, the condition reacted are as follows: room temperature Reaction 4 hours.
In an embodiment of the invention, when carrying out step 2, the methylene chloride of 2- hydroxyethyl disulfide is added dropwise Reaction condition is after solution: room temperature reaction 48 hours.
In an embodiment of the invention, when carrying out step 2, in methylene chloride and methyl alcohol mixed liquor, methylene chloride With methanol volume ratio 50:1.
In an embodiment of the invention, step 3 method particularly includes: Chlorambucil, dicyclohexyl carbon two are sub- After reaction, the 2- (2- hydroxyethyl) two obtained by step 2 is slowly added dropwise in amine, the dichloromethane solution of 4-dimethylaminopyridine Sulfenyl) ethyl -2,2- dimethyl -1,3- benzodioxole -5- ethyl propionate dichloromethane solution, reaction, with two Chloromethanes is the purification of leacheate column separation, and rotary evaporation obtains light yellow solid ((2- (2,2- dimethyl -1,3- benzos two Oxole -5- propiono) oxygroup) ethyl) -4- (4- (bis- (2- chloroethyl) amino) phenyl) methyl butyrate.
In an embodiment of the invention, when carrying out step 3,2- (2- hydroxyethyl) disulfide group) ethyl -2,2- Dimethyl -1,3- benzodioxole -5- ethyl propionate, dicyclohexylcarbodiimide, mole of 4-dimethylaminopyridine Dosage is respectively 1.2 times of Chlorambucil mole dosage.
In an embodiment of the invention, when carrying out step 3, Chlorambucil, dicyclohexylcarbodiimide, 4- The dichloromethane solution of dimethylamino naphthyridine, the condition reacted are as follows: normal-temperature reaction 4 hours.
In an embodiment of the invention, when carrying out step 3,2- (2- hydroxyethyl) disulfide group is added dropwise) ethyl- Reaction condition is after the dichloromethane solution of 2,2- dimethyl -1,3- benzodioxole -5- ethyl propionate: room temperature reaction 48 hours.
In an embodiment of the invention, step 4 method particularly includes: the ((2- (2,2- bis- that is obtained by step 3 Methyl-1,3- benzodioxole -5- propiono) oxygroup) ethyl) -4- (4- (bis- (2- chloroethyl) amino) phenyl) fourth The dichloromethane solution of sour methyl esters slowly instills trifluoroacetic acid, reaction, and rotary evaporation is dry, obtains light yellow solid benzenebutanoic acid Mustargen-dopamine conjugate molecules.
In an embodiment of the invention, when carrying out step 4, the volumetric usage of methylene chloride is generally trifluoro second 1.67 times of sour volumetric usage.
In an embodiment of the invention, when carrying out step 4, ((2- (2,2- dimethyl -1,3- benzo dioxas Cyclopentene -5- propiono) oxygroup) ethyl) and -4- (4- (bis- (2- chloroethyl) amino) phenyl) methyl butyrate quality (mg) it is general It is 100-200 times of the volume (mL) of trifluoroacetic acid.Trifluoroacetic acid is more or lacks some shadows not big for reaction process It rings, general dosage is 150-160 times.
In an embodiment of the invention, when synthesizing Chlorambucil-poly-dopamine prodrug nanoparticle, benzenebutanoic acid Mustargen-dopamine conjugate molecules quality is 1.10~4.29 times of dopamine hydrochloride quality.
In an embodiment of the invention, when synthesizing Chlorambucil-poly-dopamine prodrug nanoparticle, by mg and ML unit meter, the quality of trishydroxymethylaminomethane are 20.5 times of the volume of distilled water.
In an embodiment of the invention, when synthesizing Chlorambucil-poly-dopamine prodrug nanoparticle, when dialysis Dialysis bag retention molecular weight used is 3500, is dialysed two days, distilled water × 8 1000mL.
Chlorambucil-poly-dopamine prodrug nanoparticle synthetic route is synthesized based on the above method are as follows:
The present invention has efficiently synthesized Chlorambucil-dopamine conjugate prodrugs and its poly- DOPA of Chlorambucil- Amine prodrug nanoparticle；
In synthetic method of the present invention, anticancer drug chlorambucil is conjugated in nanoparticle, to realize in particular cancer Intracellular release drug；
Synthesizing nano-particle of the present invention has strong absorption near infrared region, can be effectively wavelength in 650nm to 1000nm Near infrared light be converted into heat kill cancer cell, thus realize photo-thermal therapy (PT) act on.At the same time, disulfide bond can be It disconnects under human body glutathione (GSH) to release drug, to realize that chemotherapy (CT) is acted on.It is treated by photo-thermal and is assisted with chemotherapy The same factor (CI) calculation formula CI=[IC50(combination CT)/IC50(CT)]+[IC50(combination PT)/IC50 (PT)] available, Chlorambucil-dopamine prodrug nanoparticle CI=0.43.CI < 1 synthesized by the present invention indicates tool There is synergistic effect.(IC50Indicate 503nhibiting concentration).Therefore, the present invention provides one kind to have near infrared absorbing, reduction response Property, chemotherapy and the integrated Biodegradable high molecular pharmaceutical carrier of light heat-therapeutic action, play collaboration effect in cancer treatment It answers.
The present invention further provides based on Chlorambucil-poly-dopamine prodrug nanoparticle antitumor application thereof.Specifically For, it is based on mild light treatment-chemotherapy combined treatment technology, the nanoparticle is used for the seamless treatment of tumour.
A kind of application of Chlorambucil-poly-dopamine prodrug nanoparticle, the nanoparticle are used to prepare anti-swollen Tumor medicine.
In a specific embodiment of the invention, it is anti-that the nanoparticle is used to prepare photo-thermal treatment-chemotherapy combined treatment Tumour medicine, wherein Chlorambucil is Chemotherapy, and poly-dopamine is light heat-therapeutic action, and the two has high Collaboration effect.
In a specific embodiment of the invention, the nanoparticle kills breast cancer cell.
In a specific embodiment of the invention, the nanoparticle is with the following functions: having Half-life in vivo long； Inherent photothermal imaging and photoacoustic imaging ability；The toy fluorescence imaging function that adriamycin drug mediates.
In a specific embodiment of the invention, the nanoparticle is after tail vein one injection and an illumination, institute The nanoparticle stated realizes breast carcinoma subcutaneous tumor seamless ablation completely after 6 days, and without tumour during treatment in 50 days Recurrence.
In a specific embodiment of the invention, the volume of the breast carcinoma subcutaneous tumor is 60-80mm3。
In a specific embodiment of the invention, the maximum temperature that an illumination generates is no more than 50 DEG C.
It is (i.e. big by light application time and hot spot that space-time manipulation may be implemented in nanoparticle of the invention in anti-tumor effect It is small and realize), reach pinpoint tumour ablation, and pass through enhancing infiltration and retention effect (EPR) (i.e. prodrug nanoparticle Son has suitable nano-scale and surface charge, to be advantageously implemented EPR effect) that prodrug nanoparticle may be implemented is passive Targeting is to tumor locus；Secondly, tumor vascular expansion and membrane permeability can be enhanced by mild photo-thermal effect Property, it is effectively facilitated the endocytosis of accumulation and infiltration and tumour cell of the nanoparticle in tumour；Finally, treat can be with for photo-thermal Inducing antitumor immunity further increases chemotherapy effect, makees to generate synergistic antitumor in photo-thermal treatment-chemotherapy combined treatment With.This mild photo-thermal treatment-chemotherapy combined treatment technology is that a new path is opened up in accurate treatment of cancer, has and potentially faces Bed application prospect.
Compared with prior art, excellent effect of the invention is as follows:
(1) Chlorambucil-poly-dopamine prodrug nanoparticle have high drug load, can in tumour cell lysosome Chlorambucil active medicine is released under acid condition and glutathione effect, realizes efficient chemotherapy effect；Again with mild light Thermotherapy combines (light intensity 1W/cm2, wavelength 808nm, light application time 10 minutes), realize mild photo-thermal treatment-change on cellular level Treat integrative coordinated therapeutic effect.
(2) Chlorambucil-poly-dopamine prodrug nanoparticle has longer blood residence time in blood, this Be conducive to prodrug nanoparticle in the enrichment of tumor locus.
(3) Chlorambucil-poly-dopamine prodrug nanoparticle have inherent photothermal imaging and photoacoustic imaging ability, And the toy fluorescence imaging function that adriamycin drug mediates, to reach the accurate tumour ablation of imaging guidance.
(4) chemotherapy and the treatment of mild photo-thermal are combined, has developed the photo-thermal treatment for the seamless treatment of tumour-chemotherapy integration Technology.
(5) the mild photo-thermal treatment-chemotherapy integrated technique is easy to operate, it is only necessary to primary intravenous injection and an illumination, just Solid tumor (volume 60-80mm can be achieved3) ablation and seamless treatment completely, there is great potential applicability in clinical practice.
(6) present invention also has near-infrared absorption, pH and redox responsiveness, photo-thermal treatment-chemotherapy one for preparation The polymeric nano medicine carrier of body provides experiment porch.
Detailed description of the invention
Fig. 1 is Chlorambucil-dopamine conjugate molecules hydrogen spectrum；
Fig. 2 is Chlorambucil-dopamine conjugate molecules carbon spectrum；
Fig. 3 is Chlorambucil-dopamine conjugate molecules mass spectrum；
Fig. 4 is Chlorambucil-poly-dopamine prodrug nanoparticle dynamic light scattering map；
Fig. 5 is Chlorambucil-poly-dopamine prodrug nanoparticle dynamic light scattering map；
Fig. 6 Chlorambucil-signal of the poly-dopamine prodrug nanoparticle to MCF-7 growth of tumour cell inhibiting effect Figure；
Fig. 7 Chlorambucil-signal of the poly-dopamine prodrug nanoparticle to MCF-7 growth of tumour cell inhibiting effect Figure；
Fig. 8 is Chlorambucil-poly-dopamine prodrug nanoparticle pharmacokinetics schematic diagram；
Fig. 9 is Chlorambucil-poly-dopamine prodrug nanoparticle small animal imaging schematic diagram；
Figure 10 is Chlorambucil-poly-dopamine prodrug nanoparticle photothermal imaging schematic diagram；
Figure 11 is Chlorambucil-poly-dopamine prodrug nanoparticle photoacoustic imaging schematic diagram；
Figure 12 is that Chlorambucil-poly-dopamine prodrug nanoparticle acts on (tumour body to MCF-7 Tumor growth inhibition Product) schematic diagram；
Figure 13 is that Chlorambucil-poly-dopamine prodrug nanoparticle acts on (Mice Body to MCF-7 Tumor growth inhibition Schematic diagram again)；
Figure 14 is that Chlorambucil-poly-dopamine prodrug nanoparticle acts on (mouse survival to MCF-7 Tumor growth inhibition Rate) schematic diagram；
Figure 15 is Chlorambucil-poly-dopamine prodrug nanoparticle to the tumor locus for being vaccinated with MCF-7 tumor-bearing mice Pathology and immunohistochemical analysis schematic diagram；
Figure 16 is Chlorambucil-poly-dopamine prodrug nanoparticle to the major organs for being vaccinated with MCF-7 tumor-bearing mice The pathology and immunohistochemical analysis schematic diagram of (heart, liver, spleen, kidney and lungs).
Wherein, in Fig. 6-Figure 16, that CB and PDA respectively refer to generation is Chlorambucil and poly-dopamine, PDCB30And PDCB40 Chlorambucil-poly-dopamine prodrug nanoparticle (content of Chlorambucil is different) are referred to, NIR refers to near-infrared laser, BSO refers to fourth methyllanthionine-sulphoxide imine, statistical significance data: P < 0.001 * * *.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The preparation of 1 Chlorambucil of embodiment-dopamine conjugate molecules
Step 1,3, the 4- dihydroxyphenyl propionic acid of 1000mg, is dissolved in the anhydrous propanone of 20mL, slowly by 383 μ L Phosphorus trichloride instilled in above-mentioned reaction flask dropwise, after being reacted 6 hours under 0 DEG C of ice-water bath, water pump rotary evaporation.Crude product is molten It in the mixture of Xie Shui and ether, is extracted using separatory funnel, organic layer is extracted 3 times with water, and concentrate drying obtains white Color powder 2,2- dimethyl -1,3- benzodioxole -5- propionic acid.Yield 60%.
Step 2,2,2- dimethyl -1,3- benzodioxole -5- propionic acid of 222mg, two hexamethylenes of 247.2mg Base carbodiimide, the 4-dimethylaminopyridine of 146.4mg are dissolved in 5mL anhydrous methylene chloride, will after room temperature reaction 4 hours The dichloromethane solution of the 2- hydroxyethyl disulfide of 180mg is instilled dropwise in above-mentioned reaction flask.After room temperature reaction 48 hours, rotation Turn evaporation.Column is filled using methylene chloride, leacheate is methylene chloride: the mixed solvent of methanol (50:1), Rf=0.36.Vacuum is dried Case is dry.Product is light yellow solid 2- (2- hydroxyethyl) disulfide group) ethyl -2,2- dimethyl -1,3- benzo dioxane Amylene -5- ethyl propionate.Yield 60%.
Step 3, the Chlorambucil of 304mg, the dicyclohexylcarbodiimide of 247.2mg, the 4- diformazan ammonia of 146.4mg Yl pyridines are dissolved in 5mL anhydrous methylene chloride, after room temperature reaction 4 hours, by 2- (2- hydroxyethyl) disulfide group of 358mg) second The dichloromethane solution of base -2,2- dimethyl -1,3- benzodioxole -5- ethyl propionate instills above-mentioned reaction flask dropwise In.After room temperature reaction 48 hours, rotary evaporation.Column is filled using methylene chloride, leacheate is dichloromethane solvent, Rf=0.32. Vacuum drying oven is dry.Product is light yellow solid ((2- (2,2- dimethyl -1,3- benzodioxole -5- propiono) oxygen Base) ethyl) -4- (4- (bis- (2- chloroethyl) amino) phenyl) methyl butyrate.Yield 80%.
Step 4, by light yellow solid ((2- (2,2- dimethyl -1,3- benzodioxole -5- propiono) oxygen Base) ethyl) -4- (4- (bis- (2- chloroethyl) amino) phenyl) methyl butyrate is dissolved in 5mL methylene chloride, it is slowly added into 3mL tri- Fluoroacetic acid is stirred at room temperature 12 hours, rotary evaporation.30 DEG C of vacuum drying ovens are dry, obtain light yellow solid Chlorambucil-DOPA Amine conjugate molecules.Yield 100%.
Chlorambucil made from the present embodiment-dopamine conjugate molecules hydrogen spectrum is as shown in Figure 1, peak position in detail is returned Belong to:1H NMR(400MHz,CD3), OD δ (ppm)=1.90 (m, 2H, CCH2CH2CH2- Ar), 2.33 (dd, J=7.6,7.2Hz, 2H,CCH2CH2CH2-Ar),2.57(m,4H,CCH2CH2CH2-Ar and CCH2CH2-Ar),2.87(m,6H,CH2SSCH2and CCH2CH2-Ar),3.60-3.68(m,4H,N(CH2CH2Cl)2,4.32(m,4H,CH2CH2SSCH2CH2),6.60(m,5H,Ar), 7.07(m,2H,Ar).
Chlorambucil made from the present embodiment-dopamine conjugate molecules carbon spectrum is as shown in Fig. 2, peak position in detail is returned Belong to:13C NMR(100MHz,CDCl3), δ (ppm)=174.88 (C1), 174.49 (C2), 144.00 (C3), 142.47 (C4), 139.97(C5),136.74(C6),133.18(C7),130.53(C8,C9),120.94(C10),116.12(C11,C12), 115.39(C13,C14),62.63(C15,C16),56.11(C17,C18),39.06(C19,C20),37.39(C21),36.23 (C22),34.36(C23),33.70(C24),30.44(C25),29.13(C26),26.56(C27).
Chlorambucil made from the present embodiment-dopamine conjugate molecules mass spectrum is as shown in Figure 3: TOF-MS:calcd, 604.1361[M+H]；found,604.1376[M+H]+.
The preparation of 2 redox responsiveness Chlorambucil of embodiment-poly-dopamine prodrug nanoparticle
410mg trishydroxymethylaminomethane is added in 50mL round-bottomed flask, 20mL distilled water, 30 DEG C are stirred 30 minutes, The Chlorambucil of 12.5mg dopamine hydrochloride and 53.7mg-dopamine conjugate molecules are dissolved in 1mL distilled water, and quickly infuse It is mapped in above-mentioned solution, the reaction time 24 hours, nano-particle solution is in black.Distilled water dialysis (dialysis bag retention molecular weight 3500) two days, distilled water × 8 1000mL.Freeze-drying 48 hours.Yield is 10%~30%.
Chlorambucil made from the present embodiment-poly-dopamine prodrug nanoparticle dynamic light scattering map such as Fig. 4 institute Show.
The preparation of 3 redox responsiveness Chlorambucil of embodiment-poly-dopamine prodrug nanoparticle
The present embodiment step with embodiment 2, the difference is that, dopamine hydrochloride and Chlorambucil-dopamine are sewed The quality of adduct molecule is respectively 12.5mg and 17.4mg.The present embodiment yield is 30%~40%.
Chlorambucil made from the present embodiment-poly-dopamine prodrug nanoparticle dynamic light scattering map such as Fig. 5 institute Show.
4 Chlorambucils of embodiment-influence of the poly-dopamine prodrug nanoparticle to breast cancer cell
The Chlorambucil being prepared using in embodiment 2-poly-dopamine prodrug nanoparticle is as making in the present embodiment Use raw material.
Prodrug nanoparticle PDCB is used in the present embodiment40(the poly- DOPA of the Chlorambucil-being prepared in embodiment 2 Amine prodrug nanoparticle, Chlorambucil concentration are respectively 40 μ g/mL), it joined fourth methyllanthionine-sulphoxide imine benzenebutanoic acid nitrogen Mustard-poly-dopamine prodrug nanoparticle, Chlorambucil are configured to Chlorambucil concentration with cell culture fluid respectively and are respectively 40, then 20,10,5,2.5,1.25,0.625,0.32 μ g/mL is cultivated 72 hours with MCF-7 cell (breast cancer), in addition, right In Chlorambucil-poly-dopamine prodrug nanoparticle, it joined the poly- DOPA of fourth methyllanthionine-sulphoxide imine Chlorambucil- Amine prodrug nanoparticle, Chlorambucil needs separately set one group respectively, after culture 12 hours, then are carried out to it with near-infrared laser Illumination 10min (808nm, 1w/cm2) continues culture 48 hours.Cell activity test is carried out using MTT method, as a result such as Fig. 6 It is shown.What abscissa CB was referred to is the concentration of Chlorambucil in Fig. 6, and in figure, CB refers to Chlorambucil experimental group, CB+NIR Refer to that Chlorambucil with after cancer cell culture 12 hours, then carries out with near-infrared laser the experimental group of illumination, PDCB to it40It is Refer to Chlorambucil-poly-dopamine prodrug nanoparticle experimental group, PDCB40+ NIR refers to Chlorambucil-poly-dopamine prodrug Nanoparticle is with after cancer cell culture 12 hours, then the experimental group of illumination, PDCB are carried out with near-infrared laser to it40+ BSO refers to It joined fourth methyllanthionine-sulphoxide imine Chlorambucil-poly-dopamine prodrug nanoparticle, PDCB40+ BSO+NIR, which refers to, to be added Enter fourth methyllanthionine-sulphoxide imine Chlorambucil-poly-dopamine prodrug nanoparticle with after cancer cell culture 12 hours, then The experimental group of illumination is carried out to it with near-infrared laser.
For MCF-7 cell, by 6 software of GraphPad Prism it can be calculated that independent photo-thermal treats (PT) in Fig. 6 503nhibiting concentration (IC50) is 69.34 μ g/mL, and the 503nhibiting concentration (IC50) of independent chemotherapy (CT) is 9.72 μ g/mL, photo-thermal The 503nhibiting concentration (IC50) for the treatment of-chemotherapy (PT-CT) combination therapy is 8.81 μ g/mL (5.23PT+3.49CT), is assisted according to formula The same factor (CI)=[IC50 (combination CT)/IC50 (CT)]+[IC50 (combination PT)/IC50 (PT)], CI=0.43 is calculated, shows that photo-thermal treatment-chemotherapy integrated technique has excellent extracorporeal anti-tumor synergistic effect.It simultaneously can also With discovery, Chlorambucil-poly-dopamine prodrug nanoparticle shows high cytotoxicity for single medicine, illustrates Chlorambucil-poly-dopamine prodrug nanoparticle shows the ability for killing cancer cell well.And it joined fourth methyllanthionine- The Chlorambucil of sulphoxide imine-poly-dopamine prodrug nanoparticle shows low cytotoxicity for single medicine, this Illustrate that fourth methyllanthionine-sulphoxide imine inhibits the generation of intracellular glutathione, thus keep cystine linkage not easy to break, release Active medicine is reduced.And for light group, it shows to compare after Chlorambucil-poly-dopamine prodrug nanoparticle illumination High cytotoxicity for after single medicine illumination further illustrates that the Chlorambucil-poly-dopamine prodrug nanoparticle exists There is good photo-thermal treatment-integrated synergistic therapeutic action of chemotherapy in terms of extracorporeal anti-tumor.
5 Chlorambucils of embodiment-influence of the poly-dopamine prodrug nanoparticle to breast cancer cell
The present embodiment step with embodiment 4, the difference is that, in Chlorambucil-poly-dopamine prodrug nanoparticle The content of Chlorambucil is different.PDCB is used in the present embodiment30(Chlorambucil-poly-dopamine prodrug nanoparticle, benzene fourth Sour mustargen concentration is respectively 30 μ g/mL), as a result as shown in Figure 7.
For MCF-7 cell, by 6 software of GraphPad Prism it can be calculated that independent photo-thermal treats (PT) in Fig. 7 503nhibiting concentration (IC50) is 69.34 μ g/mL, and the 503nhibiting concentration (IC50) of independent chemotherapy (CT) is 9.92 μ g/mL, photo-thermal The 503nhibiting concentration (IC50) for the treatment of-chemotherapy (PT-CT) combination therapy is 12.07 μ g/mL (8.45PT+3.62CT), according to above-mentioned Formula calculates CI=0.49, shows that photo-thermal treatment-chemotherapy integrated technique has good extracorporeal anti-tumor synergistic effect.Together When it has also been discovered that, Chlorambucil-poly-dopamine prodrug nanoparticle shows high cytotoxicity for single medicine, Illustrate that Chlorambucil-poly-dopamine prodrug nanoparticle shows the ability for killing cancer cell well.And it joined fourth Methyllanthionine-sulphoxide imine Chlorambucil-poly-dopamine prodrug nanoparticle shows low cell for single medicine Toxicity, this illustrates that fourth methyllanthionine-sulphoxide imine inhibits the generation of intracellular glutathione, to keep cystine linkage not easily broken It splits, the active medicine of release is reduced.And for light group, after Chlorambucil-poly-dopamine prodrug nanoparticle illumination High cytotoxicity for after single medicine illumination is shown, further illustrates the Chlorambucil-poly-dopamine prodrug Nanoparticle has good photo-thermal treatment-integrated synergistic therapeutic action of chemotherapy in terms of oncotherapy.
6 Chlorambucils of embodiment-poly-dopamine prodrug nanoparticle (PDCB40) pharmacokinetics
Pass through 500 μ L Chlorambucil of tail vein injection-poly-dopamine prodrug nanoparticle (8mg/mL) or free benzene 0.25,0.5,1,2,4,6,8,12,14 and 24 hour in butyric acid mustargen (8mg/kg) to SD rat body, and after injection is respectively Blood (0.3mL) is taken by eye frame and (10 minutes, 3000rpm) are centrifuged to harvest blood serum sample, at -20 DEG C to blood sample Then the Chlorambucil content in serum is analyzed in freezing by fluorescent spectrometry.In medicine generation, is calculated by using OriginPro 8 Kinetic parameter.
Free Chlorambucil and Chlorambucil-poly-dopamine prodrug nanoparticle are arrived by tail vein injection respectively In SD rat, and eye socket is carried out to experimental rat in regular hour point and takes blood, collected blood sample and be centrifuged to obtain upper layer Blood serum sample, and then analyze drug in blood serum content and change with time, Fig. 8 gives free Chlorambucil and benzenebutanoic acid nitrogen Mustard-poly-dopamine prodrug nanoparticle is in the intracorporal plasma metabolism situation of SD rat, and as seen from the figure, dissociate Chlorambucil Quickly, at 2 hours or so, the free Chlorambucil almost all in blood was metabolized accretion rate in blood, and benzene For butyric acid mustargen-poly-dopamine prodrug nanoparticle after injection 24 hours, the Chlorambucil in blood is still maintained at one Very high concentration, and can calculate Chlorambucil-poly-dopamine prodrug nanoparticle half-life period (t1/2 β, 1.34 0.17h) it is about 3.62 times of free Chlorambucil half-life period (0.37 0.04h), and the gross area under curve (AUC, 33.12 0.85mg.h/L) 33.8 times of about free Chlorambucil (0.98 0.02mg.h/L).It illustrates and free benzenebutanoic acid nitrogen Mustard is compared, and Chlorambucil-poly-dopamine prodrug nanoparticle has longer blood residence time in blood, and advantageous In the enrichment in tumor locus.
7 Chlorambucils of embodiment-poly-dopamine prodrug nanoparticle (PDCB40) to the adriamycin drug of breast cancer cell The toy fluorescence imaging of mediation
By tail vein to will be vaccinated with injected in MCF-7 tumor-bearing mice 200 μ L adriamycin drugloading rates be 53% benzene fourth Sour mustargen-poly-dopamine prodrug nanoparticle (1mg/mL) or free adriamycin (5.3mg/kg), and after injection 1,2,4,6, 8,10,12,14 and 24 hours using Kodak's multi-mode imaging system (adriamycin: excitation wavelength=530nm, launch wavelength= Fluorescence imaging in mouse body 600nm) is monitored, as a result as shown in Figure 9.
For the small animal imaging of MCF-7 tumor model, before Chlorambucil-poly-dopamine with adriamycin loading Mouse after the injection of medicine nanoparticle gradually increases as time increases in the fluorescence signal intensity of tumor locus, small 10 When become most strong, then gradually die down, but fluorescence signal intensity still can be observed at 24 hours；And for free adriamycin note Mouse after the penetrating fluorescence signal intensity of tumor locus in 6 hours be rapidly reached it is most strong, and sharply weakened at 24 hours to It is negligible.These statistics indicate that load Chlorambucil-poly-dopamine prodrug nanoparticle of adriamycin compared to Ah There is longer blood residence time for mycin.
8 Chlorambucils of embodiment-poly-dopamine prodrug nanoparticle (PDCB40) to the photothermal imaging of breast cancer cell
MCF-7 tumor-bearing mice will be vaccinated with and be respectively divided into seven groups: physiological saline, physiological saline+NIR, Chlorambucil (8mg/kg), poly-dopamine (2mg/mL), poly-dopamine+NIR (2mg/mL), Chlorambucil-poly-dopamine prodrug nanoparticle Son (2mg/mL), Chlorambucil-poly-dopamine prodrug nanoparticle+NIR (2mg/mL).Injection in 0th day is primary, and injects 10 Light is carried out to physiological saline+NIR and poly-dopamine+NIR, Chlorambucil-poly-dopamine prodrug nanoparticle+NIR after hour According to 10min (808nm, 1w/cm2), at the same every 30 seconds with use thermal infrared imager record mouse photothermal image and tumour portion The temperature change of position, the results are shown in Figure 10.
For the photothermal imaging of MCF-7 tumor model, near infrared light mouse tumor position after five minutes, Inject the tumor locus temperature of poly-dopamine nanoparticle or Chlorambucil-poly-dopamine prodrug nanoparticle mouse from 34.5 DEG C rise to about 45 DEG C, are then slowly heated to about 47 DEG C at 10 minutes, on the contrary, PBS group only increases about 3.8 DEG C.It please infuse Meaning, cancer cell are easy the high fever excessively by 43 DEG C or more and are killed.Chlorambucil-poly-dopamine is illustrated in these tables of data Prodrug nanoparticle can pass through the mild thermotherapy effect of generation of the near infrared light to tumor locus.
9 Chlorambucils of embodiment-poly-dopamine prodrug nanoparticle (PDCB40) to the photoacoustic imaging of breast cancer cell
By tail vein to the Chlorambucil for injecting 200 μ L in MCF-7 tumor-bearing mice-poly-dopamine prodrug will be vaccinated with Nanoparticle (1mg/mL) or poly-dopamine nanoparticle (1mg/mL), and it is 1,2,4,6,8,10,12,14 and 24 small after injection When using 128 PA tomographic system of commercialization Endra Nexus acquisition optoacoustic (PA) signal, as a result as shown in figure 11.
For the photoacoustic imaging of MCF-7 tumor model, with poly-dopamine nanoparticle (control group) and benzenebutanoic acid nitrogen Mouse after the injection of mustard-poly-dopamine prodrug nanoparticle, the photoacoustic signal of tumor locus gradually increase and 10 small after injection When reach maximum value, be with the PA signal strength of the mouse after the injection of Chlorambucil-poly-dopamine prodrug nanoparticle at this time 4.1 times of (0 hour) PA signal strength not when injecting Chlorambucil-poly-dopamine prodrug nanoparticle, but compare The PA signal strength weakening 4.5% of mouse after the injection of control group poly-dopamine nanoparticle, subsequent PA signal strength gradually subtract It is weak but 1.8 times high in signal strength of 24 hours PA signal strengths still than 0 hour.These illustrate Chlorambucil-poly-dopamine Prodrug nanoparticle has inherent and excellent photoacoustic imaging ability in vivo, it is made to can serve as controlling as the accurate cancer of guidance The means for the treatment of.
10 Chlorambucils of embodiment-poly-dopamine prodrug nanoparticle (PDCB40) influence to MCF-7 tumour growth is real It tests
MCF-7 tumor-bearing mice will be vaccinated with and be respectively divided into seven groups: physiological saline, physiological saline+NIR, Chlorambucil (8mg/kg), poly-dopamine (2mg/mL), poly-dopamine+NIR (2mg/mL), Chlorambucil-poly-dopamine prodrug nanoparticle Son (2mg/mL), Chlorambucil-poly-dopamine prodrug nanoparticle+NIR (2mg/mL).Injection in 0th day is primary, and injects 10 Light is carried out to physiological saline+NIR and poly-dopamine+NIR, Chlorambucil-poly-dopamine prodrug nanoparticle+NIR after hour Mouse weigh and measure gross tumor volume according to 10min (808nm, 1w/cm2), while every 1 day, as a result as schemed 12, shown in Figure 13.
In Figure 12, abscissa indicates that mouse receives the number of days of experiment, and ordinate indicates mouse tumor volume, and Tu12Zhong is horizontal Coordinate representation mouse receives the number of days of experiment, and ordinate indicates mouse weight.Figure 12, in 13, PBS refers to injecting normal saline pair According to group, after PBS+NIR refers to injecting normal saline 10 hours, then the experimental group of illumination is carried out with near-infrared laser to it, CB is Pointed injection Chlorambucil experimental group, PDA are pointed injection poly-dopamine experimental groups, and PDA+NIR is that pointed injection poly-dopamine 10 is small Shi Hou, then the experimental group of illumination, PDCB are carried out with near-infrared laser to it40Refer to injection Chlorambucil-poly-dopamine prodrug Nanoparticle experimental group, PDCB40+ NIR refers to injection Chlorambucil-, and poly-dopamine prodrug nanoparticle is after 10 hours, then uses Near-infrared laser carries out the experimental group of illumination to it.
For MCF-7 tumor model, Chlorambucil-poly-dopamine prodrug nanoparticle+NIR group, the 6th day all Mouse tumour is melted completely and is not scabbed, and does not occur the rebound phenomenon of tumour during 50 days for the treatment of, And (Figure 12,13) is had little effect to mouse weight.But before poly-dopamine+NIR group and Chlorambucil-poly-dopamine The tumour sustainable growth of mouse for medicine nanoparticle subgroup, but their tumor growth rate is but less than physiological saline group, life Manage salt water+NIR group, poly-dopamine group and Chlorambucil group (Figure 12).In the research of survival experiment, due to gross tumor volume Excessive (> 1500mm3), mouse in physiological saline group, physiological saline+NIR group and poly-dopamine group is at the 32-36 days without depositing It is living, and organized and none survival (figure of Chlorambucil-poly-dopamine prodrug nanoparticle subgroup mouse in the 50th day poly-dopamine+NIR 14).It is in one injection and once mild for all mouse in Chlorambucil-poly-dopamine prodrug nanoparticle+NIR group Keep seamless tumour ablation completely within 50 days after near infrared light.This show Chlorambucil-poly-dopamine prodrug nanoparticle+ NIR group produces fabulous internal synergistic therapeutic effect over the course for the treatment of.In addition, Chlorambucil-poly-dopamine prodrug is received All mouse in rice corpuscles+NIR group weight in 50 days has almost no change, and shows Chlorambucil-poly-dopamine prodrug Nanoparticle does not have toxicity (Figure 13).
11 Chlorambucils of embodiment-poly-dopamine prodrug nanoparticle (PDCB40) to the pathology of MCF-7 tumour and exempt from Epidemic disease histochemical analysis
At the 32nd day, every group of mouse in embodiment 10 is killed one, dissects tumour and major organs (heart, liver It is dirty, spleen, lung and kidney), it is washed with brine, weighs, takes pictures and be fixed for histology, TUNEL and PCNA survey with 4% formaldehyde It is fixed.
The tumour of excision and major organs (liver, kidney, spleen, lung and heart) are subjected to histologic analysis (figure with H&E dyeing 15A and Figure 16).With Chlorambucil group, poly-dopamine+NIR group and Chlorambucil-poly-dopamine prodrug nanoparticle subgroup Tumour is compared, and observes that the quantity of cell is seriously reduced in Chlorambucil-poly-dopamine prodrug nanoparticle+NIR group, and core is received The phenomenon that contracting and division.Poly-dopamine+NIR group and Chlorambucil-poly-dopamine prodrug nanoparticle subgroup and Chlorambucil- The major organs that poly-dopamine prodrug nanoparticle+NIR organizes all mouse do not have apparent Pathologic changes and a damage, but benzene The heart of butyric acid mustargen group, spleen and kidney, which have, slightly to be damaged.In addition, TUNEL (Figure 15 B) and PCNA (Figure 15 C) are respectively intended to Detect apoptosis of tumor cells and proliferation.With Chlorambucil group, poly-dopamine+NIR group and Chlorambucil-poly-dopamine prodrug The tumour of nanoparticle subgroup is compared, and is shown in Chlorambucil-poly-dopamine prodrug nanoparticle+NIR group apparent thin Born of the same parents' apoptosis has a small amount of Apoptosis in poly-dopamine group, and Apoptosis is minimum in physiological saline and physiological saline+NIR group.Phase Instead, positive tumor cell is reduced at most in Chlorambucil-poly-dopamine prodrug nanoparticle+NIR group, followed by benzenebutanoic acid nitrogen Mustard group, poly-dopamine+NIR group and Chlorambucil-poly-dopamine prodrug nanoparticle subgroup, poly-dopamine group, physiological saline and Physiological saline+NIR group reduces minimum.Show that Chlorambucil-poly-dopamine prodrug nanoparticle does not have toxicity.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention Within protection scope.

Claims (10)

1. a kind of application of Chlorambucil-poly-dopamine prodrug nanoparticle, which is characterized in that the nanoparticle is for making Standby anti-tumor drug.
2. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 1, which is characterized in that described Nanoparticle is used to prepare the seamless therapeutic agent of tumour.
3. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 1, which is characterized in that described Nanoparticle has characteristic used below: cooperating illumination to use after injection.
4. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 1, which is characterized in that described Nanoparticle is with the following functions:
Half-life in vivo is long,
Inherent photothermal imaging and photoacoustic imaging ability,
The toy fluorescence imaging function that adriamycin drug mediates.
5. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 1, which is characterized in that described Nanoparticle is with the following functions:
After tail vein one injection and an illumination, it is complete after 6 days that the nanoparticle realizes breast carcinoma subcutaneous tumor Seamless ablation, and without tumor recurrence during treatment in 50 days.
6. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 5, which is characterized in that described The maximum temperature that illumination generates is no more than 50 DEG C.
7. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 1, which is characterized in that irradiation When, light intensity 1W/cm2, wavelength 808nm, light application time 10 minutes.
8. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 1, which is characterized in that described Nano particle structure formula is as follows:
Wherein, n, m, o are positive integer, and n, m, o are identical or not identical.
9. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 8, which is characterized in that with benzene Butyric acid mustargen-dopamine conjugate molecules are raw material, under alkaline condition with dopamine hydrochloride, in distilled water and trihydroxy methyl The in the mixed solvent of aminomethane is reacted, and dialysis obtains Chlorambucil-poly-dopamine prodrug nanoparticle；
The Chlorambucil-dopamine conjugate synthetic method, comprising the following steps:
Step 1,3,4- dihydroxyphenyl propionic acids are reacted with phosphorus trichloride, prepare 2,2- dimethyl -1,3- benzo dioxane penta Alkene -5- propionic acid；
Step 2,2,2- dimethyl -1,3- benzodioxole -5- propionic acid, dicyclohexylcarbodiimide, 4- dimethylamino Pyridine reaction adds the reaction of 2- hydroxyethyl disulfide, obtains 2- (2- hydroxyethyl) disulfide group) ethyl -2,2- dimethyl - 1,3- benzodioxole -5- ethyl propionate；
Step 3, Chlorambucil, dicyclohexylcarbodiimide, 4-dimethylaminopyridine reaction, adds 2- (2- hydroxyl second Base) disulfide group) ethyl -2,2- dimethyl -1,3- benzodioxole -5- ethyl propionate, is reacted, obtains ((2- (2,2- dimethyl -1,3- benzodioxole -5- propiono) oxygroup) ethyl) -4- (4- (bis- (2- chloroethyl) amino) Phenyl) methyl butyrate；
Step 4, ((2- (2,2- dimethyl -1,3- benzodioxole -5- propiono) oxygroup) ethyl) (4- is (double by -4- (2- chloroethyl) amino) phenyl) methyl butyrate reacts with trifluoroacetic acid, obtain Chlorambucil-dopamine conjugate molecules.
10. Chlorambucil-poly-dopamine prodrug nanoparticle application according to claim 1, which is characterized in that described Tumour is MCF-7 tumour.
CN201810960221.1A 2018-08-22 2018-08-22 Chlorambucil-poly-dopamine prodrug nanoparticle antitumor application thereof Pending CN109010827A (en)

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