CN105148279B - Dextran nano-medicament carrier and preparation method thereof and antineoplastic and preparation method thereof - Google Patents
Dextran nano-medicament carrier and preparation method thereof and antineoplastic and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of dextran nano-medicament carrier and preparation method thereof and antineoplastic and preparation method thereof.The dextran nano-medicament carrier includes the dextran nano-particle of carboxylated, and the particle diameter of the dextran nano-particle of the carboxylated is 20~100 nanometers.The carboxylated dextran nano-particle that particle diameter is 20~100 nanometers has good biological pardon and degradability, it can be used for carrying medicament and act on human body, and due to dextran the design feature of carboxylated in itself, hydrazone key carrying medicament can be passed through, hydrazone key is sensitive to pH, is broken and Targeting delivery medicine under specific pH environment.Therefore, above-mentioned dextran nano-medicament carrier can be used for the medicine-releasing system for establishing pH responses, to improve curative effect, reduce side effect.
Description
Technical field
The present invention relates to pharmaceutical technology field, more particularly to a kind of dextran nano-medicament carrier and preparation method thereof
With antineoplastic and preparation method thereof.
Background technology
At present, cancer has become the primary enemy for threatening human life and health.Traditional cancer therapy drug, such as adriamycin
Deng because it lacks specific tumor targets, it is impossible to distribution of specific is in normal cell and tumour cell, not only weak curative effect,
And the side effect of adriamycin drug dose correlation is obvious.
Under normal physiological conditions, the pH of human body different parts is different, and under pathological condition, pH changes are particularly evident, profit
The medicine-releasing system that can design pH responses with this change, so as to improve curative effect, reduces side effect to improve the targeting of medicine.
The content of the invention
Based on this, it is necessary to a kind of dextran nano-medicament carrier is provided, for establishing the medicine-releasing system of pH responses, with
Curative effect is improved, reduces side effect.
A kind of dextran nano-medicament carrier, include the dextran nano-particle of carboxylated, the right side of the carboxylated
The particle diameter for revolving sugared acid anhydride nano-particle is 20~100 nanometers.
A kind of preparation method of dextran nano-medicament carrier, comprises the following steps：
In protective gas atmosphere, dextran is dissolved in solvent, the first solution is prepared；
In protective gas atmosphere, ammonium ceric nitrate is dissolved in nitric acid, the second solution is prepared；
In protective gas atmosphere, second solution is injected in first solution, and 5 are reacted at 20~30 DEG C
~10 minutes, acrylic acid is added, is reacted 20~30 minutes at 20~30 DEG C, addition N, N- methylene-bisacrylamides, so
After continuing reaction 4~6 hours at 20~30 DEG C, isolate and purify, obtain the dextran nano-medicament carrier, the dextrorotation
Sugared acid anhydride nano-medicament carrier includes the dextran nano-particle of carboxylated, the dextran Nano medication particle of the carboxylated
Particle diameter be 20~100 nanometers.
In one of the embodiments, the method that isolates and purifies is：The pH of reaction solution is adjusted to neutrality, then will be anti-
Answer liquid to be fitted into bag filter, dialyse 72 hours in deionized water, freeze-drying, obtain the dextran Nano medication and carry
Body.
A kind of antineoplastic, including one kind in adriamycin and cinnamic acid and dextran as claimed in claim 1
Nano-medicament carrier, one kind in the adriamycin and cinnamic acid are connected to the dextran nano-medicament carrier by hydrazone key
On.
A kind of antineoplastic, including one kind, EGFR antibody and above-mentioned dextran nanometer in adriamycin and cinnamic acid
Pharmaceutical carrier, the EGFR antibody are connected with the dextran nano-medicament carrier, and one in the adriamycin and cinnamic acid
Kind is connected on the dextran nano-medicament carrier by hydrazone key.
In one of the embodiments, a kind of mass percent in the adriamycin and cinnamic acid is 23%.
A kind of preparation method of antineoplastic, comprises the following steps：
EGFR antibody and above-mentioned dextran nano-medicament carrier are mixed, and adds carbodiimide and N- hydroxysuccinimidyls acyl Asia
Amine obtains the first mixed liquor, and the pH value of first mixed liquor is adjusted into 4.5~5, reacts 2~4 hours at room temperature, separation
Purifying obtains intermediate product；
The intermediate product is dissolved in the solution containing intermediate product is obtained in solvent, contain the molten of intermediate product by described
Drop is added in hydrazine solution, and adds carbodiimide and n-hydroxysuccinimide obtains the second mixed liquor, by described second
Mixed liquor isolated and purified to obtain amido modified intermediate product in room temperature reaction 20~25 hours；
The amido modified intermediate product is dissolved in solvent, the one kind added in adriamycin and cinnamic acid, 35~
Vibrated 48~72 hours at 40 DEG C, the antineoplastic is obtained after isolating and purifying.
In one of the embodiments, the mol ratio of the EGFR antibody and the dextran nano-medicament carrier is 20
~30:1.
In one of the embodiments, it is described and add carbodiimide and n-hydroxysuccinimide obtains the first mixed liquor
The step of in, the mol ratio of the carbodiimide and n-hydroxysuccinimide is 5~10:1.
In one of the embodiments, it is described and add carbodiimide and n-hydroxysuccinimide obtains the second mixed liquor
The step of in, the mol ratio of the carbodiimide and n-hydroxysuccinimide is 5~10:1.
The carboxylated dextran nano-particle that particle diameter is 20~100 nanometers has good biological pardon and degraded
Property, it can be used for carrying medicament and act on human body, and due to dextran the design feature of carboxylated in itself, can pass through
Hydrazone key carrying medicament, hydrazone key is sensitive to pH, is broken and Targeting delivery medicine under specific pH environment.Therefore, above-mentioned dextrose
Acid anhydride nano-medicament carrier can be used for the medicine-releasing system for establishing pH responses, to improve curative effect, reduce side effect.
Brief description of the drawings
Fig. 1 is the flow chart of the preparation method of the dextran nano-medicament carrier of an embodiment；
Fig. 2 is the flow chart of the preparation method of the above-mentioned antineoplastic of an embodiment；
Fig. 3 is the synthesis schematic diagram of the preparation method of the antineoplastic shown in Fig. 2；
Fig. 4 is the TEM figures of the dextran nano-medicament carrier prepared by embodiment 1；
Fig. 5 is that releases of the DOX of the EGFR-Dextran-DOX antineoplastics of embodiment 5 under condition of different pH is bent
Line；
Fig. 6 (a)~Fig. 6 (c) is that the confocal scan microscope of the EGFR-Dextran-DOX antineoplastics of embodiment 5 is glimmering
Light colour developing figure；
Fig. 7 is antitumor for the Dextran-DOX antineoplastics of embodiment 4 and the EGFR-Dextran-DOX of embodiment 5
The antitumor curative effect experimental result of medicine.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, below in conjunction with the accompanying drawings to the present invention
Embodiment be described in detail.Many details are elaborated in the following description in order to fully understand this hair
It is bright.But the invention can be embodied in many other ways as described herein, those skilled in the art can be not
Similar improvement is done in the case of running counter to intension of the present invention, therefore the present invention is not limited to the specific embodiments disclosed below.
A kind of dextran nano-medicament carrier, is expressed as Dextran.The Dextran receives including carboxylated dextran
Rice corpuscles, the particle diameter of the dextran nano-particle of the carboxylated is 20~100 nanometers.
Dextran has good biological pardon and degradability, without observable toxicity, during its Peripheral Circulation
Between it is longer, when entering human body using the dextran nano-medicament carrier carrying medicament, security is higher.Can thus use should
Dextran nano-medicament carrier carrying medicament acts on human body.
Also, can be by hydrazone key carrying medicament due to dextran the design feature of carboxylated in itself, hydrazone key is to pH
Sensitivity, it is broken and Targeting delivery medicine under specific pH environment.Therefore, above-mentioned dextran nano-medicament carrier, Ke Yiyong
In the medicine-releasing system for establishing pH responses, to improve curative effect, side effect is reduced.
The dextran of carboxylated is easy to be modified, and being provided for dextran nano-medicament carrier load multi-medicament can
Energy.Meanwhile the nano-particle that particle diameter is 20~100nm can avoid, by the phagocyte removing of reticuloendothelial system (RES), passing through
Appropriate surface modification, it can also effectively extend the blood circulation time of dextran nano-particle, so as to improve curative effect.
Referring to Fig. 1, the preparation method of above-mentioned dextran nano-medicament carrier comprises the following steps：
Step S110：In protective gas atmosphere, dextran is dissolved in solvent, the first solution is prepared.
Protective gas can be nitrogen or inert gas, preferably nitrogen.
Dextran is the dextran that mean molecule quantity is 20000~40000.Solvent is deionized water.Dextran
Concentration in the first solution is preferably 0.05mg/mL.
Step S120：In protective gas atmosphere, ammonium ceric nitrate is dissolved in nitric acid, the second solution is prepared.
Protective gas can be nitrogen or inert gas, preferably nitrogen.
Nitric acid is 0.1N dust technology.Concentration of the ammonium ceric nitrate in the second solution is preferably 0.25mg/mL.
Step S130：In protective gas atmosphere, the second solution is injected in the first solution, and is reacted at 20~30 DEG C
5~10 minutes, acrylic acid is added, is reacted 20 minutes at 20~30 DEG C, add N, N- methylene-bisacrylamides, Ran Houyu
Continue reaction at 20~30 DEG C 4~6 hours, isolate and purify, obtain dextran nano-medicament carrier, dextran Nano medication
Carrier includes the dextran nano-particle of carboxylated, and the particle diameter of the dextran Nano medication particle of carboxylated is 20~100
Nanometer.
Protective gas can be nitrogen or inert gas, preferably nitrogen.
Second solution is injected in the first solution, to trigger free radical on dextran chain.First solution and second molten
The volume ratio of liquid is preferably 30:1~40:1.
The volume ratio of acrylic acid and the first solution is preferably 1:20~30, N, N- methylene-bisacrylamide and the first solution
Solid-to-liquid ratio be 1g:15~20mL.
The method isolated and purified is specially：The pH of reaction solution is adjusted to neutrality, then reaction solution is fitted into bag filter,
Dialyse 72 hours in deionized water, to remove unreacted monomer, be freeze-dried after having dialysed, obtain dextran nanometer medicine
Thing carrier, the dextran nano-medicament carrier include the dextran nano-particle of carboxylated, and the dextran of carboxylated is received
The particle diameter of rice drug particle is 20~100 nanometers.
Carboxylated is carried out while the preparation method of above-mentioned dextran nano-medicament carrier is by dextran nanosizing, is made
The standby dextran nano-medicament carrier for obtaining can be used for establishing the medicine-releasing system of pH responses, technique is simple, and it is low to prepare cost.
A kind of antineoplastic, including one kind in adriamycin and cinnamic acid and above-mentioned dextran nano-medicament carrier,
One kind in adriamycin and cinnamic acid is connected on dextran nano-medicament carrier by hydrazone key.
Hydrazone key is sensitive to pH, is only broken under the conditions of specific pH, the fracture release adriamycin or cinnamic acid of hydrazone key, makes
Adriamycin or cinnamic acid Targeting delivery under particular circumstances are obtained, is advantageous to improve curative effect, reduces side effect.
The preparation method of above-mentioned antineoplastic comprises the following steps：
Step S210：Above-mentioned dextran nano-medicament carrier, ethanol and the concentrated sulfuric acid are mixed to get mixed liquor, this is mixed
Close liquid to flow back 3~4 hours, saturated sodium carbonate is added after cooling and is neutralized, then add the hydrazine hydrate that mass concentration is 80%, continue
Backflow 10~20 hours, filtering, collect solid product.
The concentrated sulfuric acid is the sulfuric acid that mass percent is 85%.Dextran nano-medicament carrier, ethanol and the concentrated sulfuric acid are consolidated
Liquor ratio is preferably 1g:5mL:10mL.
The solid-to-liquid ratio of dextran nano-medicament carrier and the hydrazine hydrate that mass concentration is 80% is preferably 1g:5mL.
Step S220：Solid product is soluble in water, and adriamycin or cinnamic acid are added, then vibration 72 is small at 37 DEG C
When, dialysed 72 hours in deionized water, be freeze-dried, obtain antineoplastic.
The preparation method of above-mentioned antineoplastic generates hydrazine key first on dextran nano-medicament carrier, then hydrazine key
Hydrazone key is generated with the carbonyl reaction on adriamycin or cinnamic acid, so as to which adriamycin or cinnamic acid are carried on into dextrose by hydrazone key
On acid anhydride nano-medicament carrier, the antineoplastic that there is pH to respond is obtained.
Further, another antineoplastic is also provided, including one kind in adriamycin and cinnamic acid, EGFR (epidermises
Growth factor receptors) antibody and above-mentioned dextran nano-medicament carrier.Wherein, EGFR antibody carries with dextran Nano medication
Body phase connects, and one kind in adriamycin and cinnamic acid is connected on dextran nano-medicament carrier by hydrazone key.
EGF-R ELISA (epidermal growth factor receptor, EGFR) mutation, imbalance or mistake
Many epithelial malignancies are expressed in, are played an important role in the growth of tumour and atomization.EGFR transduces in tumor signal
Approach, regulation cell propagation, survive and played an important role in atomization.For human normal tissue,
The EGFR of high expression is can detect in about 60% triple negative breast cancer (TNBC) patient tumors cell, thus EGFR is TNBC reasons
The therapy target thought.
For above-mentioned antineoplastic just using EGFR as action target spot, Dextran is pharmaceutical carrier, realizes that the targeting of cancer is controlled
Treat, so as to improve curative effect, reduce side effect.
Preferably, in above-mentioned antineoplastic, the mass percent of adriamycin or cinnamic acid is 23%.
Fig. 2 and Fig. 3 are referred to, the preparation method of above-mentioned antineoplastic comprises the following steps：
Step S310：EGFR antibody and above-mentioned dextran nano-medicament carrier are mixed, and adds carbodiimide and N- hydroxyls
Base succinimide obtains the first mixed liquor, and the pH value of the first mixed liquor then is adjusted into 4.5~5, at room temperature react 2~
4 hours, isolate and purify to obtain intermediate product.EGFR antibody is EGFR monoclonal antibody.The mol ratio of EGFR antibody and Dextran
For 20~30:1, preferably 25:1.
The mol ratio of carbodiimide (EDAC) and n-hydroxysuccinimide (NHS) is preferably 5~10:1.Revolve sugared acid anhydride nanometer
The mol ratio of pharmaceutical carrier Dextran and carbodiimide is 1:50~100.
The pH value of the first mixed liquor is adjusted to 4.5~5 using MES buffer solutions.PH value is 4.5~5 the first mixed liquor
React 2 hours at room temperature, isolate and purify to obtain intermediate product, intermediate product is EGFR antibody and above-mentioned dextran nanometer medicine
The material that thing carrier is connected to form, is expressed as EGFR-Dextran.
The method isolated and purified is to be washed three times with MES buffer solutions.
The step of EGFR antibody also marks including nir dye Cy5.5 and fluorescence probe before use and quantifies EGFR.
Step S320：Intermediate product is dissolved in the solution containing intermediate product is obtained in solvent, intermediate product will be contained
Solution is added dropwise in hydrazine solution, and adds carbodiimide and n-hydroxysuccinimide obtains the second mixed liquor, and second is mixed
Liquid is closed in room temperature reaction 20~25 hours, isolates and purifies to obtain amido modified intermediate product.
Solvent is preferably anhydrous dimethyl sulphoxide (DMSO).In solution containing intermediate product, the concentration of intermediate product is excellent
Elect 0.1~0.2g/mL as.
The volume ratio of solution and hydrazine solution containing intermediate product is preferably 3:2.Carbodiimide (EDAC) and N- hydroxyls
The mol ratio of succinimide (NHS) is preferably 5~10:1.
In step S320 ,-the COOH on Dextran is reacted with hydrazine, and amino is introduced on Dextran.
The method isolated and purified is：With methanol extraction, then it is freeze-dried.Amido modified intermediate product is expressed as amino
The EGFR-Dextran of modification.
Step S330：Amido modified intermediate product is dissolved in solvent, adriamycin is added, vibrates 48 at 35~40 DEG C
~72 hours, antineoplastic is obtained after isolating and purifying.
Solvent is preferably anhydrous dimethyl sulphoxide (DMSO).
The method isolated and purified is to be dialysed 48 hours with deionized water, is then freeze-dried.
Adriamycin (DOX) is connected with amido modified EGFR-Dextran by hydrazone key, generation antineoplastic EGFR-
Dextran-DOX。
The preparation method of above-mentioned antineoplastic first synthesizes EGFR-Dextran, then carries out amino to EGFR-Dextran
Modification generates amido modified EGFR-Dextran, and amido modified EGFR-Dextran is connected with DOX by hydrazone key, generates
EGFR-Dextran-DOX.This method technique is simple, and it is low to prepare cost.
It is expanded on further below by way of specific embodiment.
Embodiment 1
Prepare dextran nano-medicament carrier
1st, in nitrogen atmosphere, the dextran that 2.5mg mean molecule quantities are 20000 is dissolved in 50mL in three-neck flask
In deionized water, stirring makes it fully dissolve, and the first solution is prepared；
2nd, in nitrogen atmosphere, 0.31g ammonium ceric nitrates are dissolved in 1.25mL 0.1N nitric acid, it is molten is prepared second
Liquid；
3rd, in protective gas atmosphere, the second solution is injected in the first solution, and after being reacted 5 minutes at 30 DEG C, so
2mL acrylic acid is added afterwards, is reacted 20 minutes at 30 DEG C, 2.75g N, N- methylene-bisacrylamides is added, then in 30
Continue reaction 4 hours at DEG C, reaction adds 1N NaOH regulation systems pH to neutrality, finally loads reaction solution and dialyse after terminating
In bag, dialyse 72 hours in deionized water, freeze-drying, obtain dextran nano-medicament carrier, the dextran nanometer
Pharmaceutical carrier includes the dextran nano-particle of carboxylated.Use dynamic light scattering method (DLS) and transmission electron microscope
(TEM) particle size is measured, TEM figures are shown in Fig. 4, and the particle diameter of the dextran nano-particle of carboxylated is 95 nanometers.
Embodiment 2
Prepare dextran nano-medicament carrier
1st, in nitrogen atmosphere, the dextran that 3.0mg mean molecule quantities are 40000 is dissolved in 60mL in three-neck flask
In deionized water, stirring makes it fully dissolve, and the first solution is prepared；
2nd, in nitrogen atmosphere, 0.6g ammonium ceric nitrates are dissolved in 2.5mL 0.1N nitric acid, the second solution is prepared；
3rd, in protective gas atmosphere, the second solution is injected in the first solution, and after being reacted 10 minutes at 20 DEG C, so
2mL acrylic acid is added afterwards, after being reacted 20 minutes at 20 DEG C, adds 2.75g N, N- methylene-bisacrylamides, Ran Houyu
Continue reaction 6 hours at 20 DEG C, reaction adds 1N NaOH regulation systems pH to neutrality, finally loaded reaction solution saturating after terminating
Analyse in bag, dialyse 72 hours in deionized water, freeze-drying, obtain dextran nano-medicament carrier, the dextran is received
Rice pharmaceutical carrier includes the dextran nano-particle of carboxylated.It is micro- using dynamic light scattering method (DLS) and transmission electron
Mirror (TEM) measures particle size, and the particle diameter of the dextran nano-particle of carboxylated is 98 nanometers.
Embodiment 3
Prepare dextran nano-medicament carrier
1st, in nitrogen atmosphere, the dextran that 3.0mg mean molecule quantities are 40000 is dissolved in 60mL in three-neck flask
In deionized water, stirring makes it fully dissolve, and the first solution is prepared；
2nd, in nitrogen atmosphere, 0.6g ammonium ceric nitrates are dissolved in 2.5mL 0.1N nitric acid, the second solution is prepared；
3rd, in protective gas atmosphere, the second solution is injected in the first solution, and after being reacted 8 minutes at 25 DEG C, so
2mL acrylic acid is added afterwards, after being reacted 20 minutes at 25 DEG C, adds 2.75g N, N- methylene-bisacrylamides, Ran Houyu
Continue reaction 5 hours at 25 DEG C, reaction adds 1N NaOH regulation systems pH to neutrality, finally loaded reaction solution saturating after terminating
Analyse in bag, dialyse 72 hours in deionized water, freeze-drying, obtain dextran nano-medicament carrier, the dextran is received
Rice pharmaceutical carrier includes the dextran nano-particle of carboxylated.It is micro- using dynamic light scattering method (DLS) and transmission electron
Mirror (TEM) measures particle size, and the particle diameter of the dextran nano-particle of carboxylated is 50 nanometers.
Embodiment 4
Prepare Dextran-DOX antineoplastics
1st, dextran nano-medicament carrier, 5mL ethanol and the 10mL concentrated sulfuric acids made from 1g embodiments 1 are mixed to get mixed
Liquid is closed, by the mixed-liquor return 4 hours, it was 7 that saturated sodium carbonate to the pH value of system is added after cooling, then adds 5mL mass
Concentration is 80% hydrazine hydrate, continues backflow 12 hours, filtering, collects solid product；
2nd, it is solid product is soluble in water, and 5g adriamycins are added, then vibrated 72 hours at 37 DEG C, in deionized water
Middle dialysis 72 hours, freeze-drying, obtains Dextran-DOX antineoplastics.
Embodiment 5
Prepare EGFR-Dextran-DOX antineoplastics
1st, the EGFR monoclonal antibody of SANT CRUZE companies of the U.S. is bought, is visited using nir dye Cy5.5 and fluorescence
Pin marks and quantifies EGFR, the dextran nanometer medicine that will be marked and prepared by fixed measured EGFR monoclonal antibody and embodiment 1
Thing carrier Dextran in molar ratio 25:1 is mixed, and then adds carbodiimide and n-hydroxysuccinimide obtains first
Mixed liquor, wherein, the mol ratio of carbodiimide and n-hydroxysuccinimide is 5:1, revolve sugared acid anhydride nano-medicament carrier Dextran
Mol ratio with carbodiimide is 1:50；The pH that the first mixed liquor is adjusted with MES buffer solutions is 4.5, then reacts 2 at room temperature
Hour, MES buffer solutions wash 3 times, obtain intermediate product；
The 2nd, intermediate product is dissolved in the solution containing intermediate product for obtaining that concentration is 0.1g/mL in anhydrous DMSO, will contained
The solution for having intermediate product is added dropwise in hydrazine solution, and adds mol ratio as 5:1 carbodiimide and N- hydroxysuccinimidyls acyl is sub-
Amine obtains the second mixed liquor, wherein, the volume ratio of solution and hydrazine solution containing intermediate product is 3:2；By the second mixed liquor
React 24 hours at room temperature, after reaction terminates, with methanol extraction, the intermediate product for being freeze-dried amido modified；
3rd, amido modified intermediate product is dissolved in anhydrous DMSO, adds adriamycin, vibrated 72 hours at 37 DEG C,
Dialysed 48 hours in deionized water, be freeze-dried, obtain EGFR-Dextran-DOX antineoplastics；
4th, Nanosurface electric charge is detected using Ventral medulla, high performance liquid chromatography (HPLC)-fluorescence detection is determined for DOX
Amount.HPLC parameters are as follows：
Detector：2487 binary channels UV-detectors, Detection wavelength 254nm；Chromatographic column：C18 posts (Dalian Yi Lite
ODS2 analytical columns, 250I build X4.6iTlm, 5 capes), stainless steel column；Guard column：Féraud door C18 (4mnl × 4mm)；Constant temperature：35
℃.Flow velocity：1.0mL/min；Mobile phase：(volume ratio of water and methanol is 28 to the mixed liquor of water and methanol:72)；Sample introduction：Wate
The pump of agate 1525,717 Autosamplers, sample size go out for 20；Work station：Breeze chromatographic work stations.
Test result indicates that DOX carrying drug ratios are up to mass percents of 77%, the DOX in EGFR-Dextran-Dox
23%.
5th, the pH response characteristics of EGFR-Dextran-DOX antineoplastics
20 milliliters of EGFR-Dextran-DOX solution is placed in dialysis and immerses 480 milliliters of pH (4,5,6,7) bufferings
In liquid, the sample that pH is respectively 4,5,6 and 7 is prepared, in selected time interval at 37 DEG C, takes outer 1 milliliter molten of bag filter
Liquid detects DOX content by HPLC, and drug release patterns are made, and wherein HPLC parameter is the same as embodiment 4.
Experimental result as shown in figure 5, as shown in Figure 5, EGFR-Dextran-DOX antineoplastics have pH response characteristics,
Regulation pH value can reach the purpose of control DOX releases.Because the pH value of peripheral circulation blood is 7~7.4, and tumor by local and tumour
Between intracellular pH value is 4~6, thus EGFR-Dextran-DOX can keep relative stabilization in blood, ensure that big portion
Divide medicine to be discharged in tumor by local and play targeted therapy effect, and reduce side effect.
6th, DOX positions and monitored in real time insoluble drug release in intracellular EGFR-Dextran-DOX antineoplastics
DOX in EGFR-Dextran-DOX antineoplastics is observed using confocal scan microscope fluorescence developing.
Experimental result such as Fig. 6 (a), Fig. 6 (b) and Fig. 6 (c) are shown, and EGFR shows green fluorescence, and DOX shows red fluorescence,
Yellow is shown as after overlapping.
From Fig. 6 (a), Fig. 6 (b) and Fig. 6 (c), the DOX in EGFR-Dextran-DOX mediates intracellular through EGFR
Medicine can be discharged after gulping down in intracellular and then reached its target spot and played CDCC.
7th, the antitumor curative effect of EGFR-Dextran-DOX and Dextran-DOX antineoplastics
From lotus people's TNBC tumour cells MDA-MB231 (the high expression of EGFR) and MDA-MB231 (EGFR knockouts) nude mice
For animal model, 3 mouse/groups, drug concentration:1 mg kg of body weight, subcutaneous administrations, 2 times/week, treat 3 weeks, see
Dextran-DOX (prepared by embodiment 3) and EGFR-Dextran-DOX anti-knurl curative effect is examined, is made with PBS and DOX
For control.
Experimental result as shown in fig. 7, give EGFR-Dextran-DOX nano-particles treatment mouse, tumor volume compared with
Dextran-DOX and non-targeted DOX nanoparticles are obviously reduced, and Dextran-DOX effect is better than non-targeted DOX.Illustrate low
Under concentration (1 mg kg of body weight), EGFR-Dextran-DOX anti-tumor activity is received compared with Dextran-DOX and non-target tropism DOX
Rice significantly increases, and Dextran-DOX antitumor activity is higher than non-target tropism DOX.
Embodiment 6
Prepare antineoplastic
1st, the EGFR monoclonal antibody of SANT CRUZE companies of the U.S. is bought, is visited using nir dye Cy5.5 and fluorescence
Pin marks and quantifies EGFR, the dextran nanometer medicine that will be marked and prepared by fixed measured EGFR monoclonal antibody and embodiment 1
Thing carrier Dextran in molar ratio 25:1 is mixed, and then adds carbodiimide and n-hydroxysuccinimide obtains first
Mixed liquor, wherein, the mol ratio of carbodiimide and n-hydroxysuccinimide is 10:1, revolve sugared acid anhydride nano-medicament carrier
The mol ratio of Dextran and carbodiimide is 1:100；The pH that the first mixed liquor is adjusted with MES buffer solutions is 5, then in room temperature
Lower reaction 4 hours, MES buffer solutions wash 3 times, obtain intermediate product；
The 2nd, intermediate product is dissolved in the solution containing intermediate product for obtaining that concentration is 0.2g/mL in anhydrous DMSO, will contained
The solution for having intermediate product is added dropwise in hydrazine solution, and adds mol ratio as 10:1 carbodiimide and N- hydroxysuccinimidyls acyl is sub-
Amine obtains the second mixed liquor, wherein, the volume ratio of solution and hydrazine solution containing intermediate product is 3:2；By the second mixed liquor
React 20 hours at room temperature, after reaction terminates, with methanol extraction, the intermediate product for being freeze-dried amido modified；
3rd, amido modified intermediate product is dissolved in anhydrous DMSO, adds cinnamic acid, vibrated 48 hours at 40 DEG C,
Dialysed 48 hours in deionized water, be freeze-dried, obtain antineoplastic.
Embodiment 7
Prepare EGFR-Dextran-DOX antineoplastics
1st, the EGFR monoclonal antibody of SANT CRUZE companies of the U.S. is bought, is visited using nir dye Cy5.5 and fluorescence
Pin marks and quantifies EGFR, the dextran nanometer medicine that will be marked and prepared by fixed measured EGFR monoclonal antibody and embodiment 1
Thing carrier Dextran in molar ratio 25:1 is mixed, and then adds carbodiimide and n-hydroxysuccinimide obtains first
Mixed liquor, wherein, the mol ratio of carbodiimide and n-hydroxysuccinimide is 8:1, revolve sugared acid anhydride nano-medicament carrier Dextran
Mol ratio with carbodiimide is 1:80；The pH that the first mixed liquor is adjusted with MES buffer solutions is 5, and then reaction 3 is small at room temperature
When, MES buffer solutions wash 3 times, obtain intermediate product；
The 2nd, intermediate product is dissolved in the solution containing intermediate product for obtaining that concentration is 0.2g/mL in anhydrous DMSO, will contained
The solution for having intermediate product is added dropwise in hydrazine solution, and adds mol ratio as 8:1 carbodiimide and N- hydroxysuccinimidyls acyl is sub-
Amine obtains the second mixed liquor, wherein, the volume ratio of solution and hydrazine solution containing intermediate product is 3:2；By the second mixed liquor
React 25 hours at room temperature, after reaction terminates, with methanol extraction, the intermediate product for being freeze-dried amido modified；
3rd, amido modified intermediate product is dissolved in anhydrous DMSO, adds adriamycin, vibrated 60 hours at 40 DEG C,
Dialysed 48 hours in deionized water, be freeze-dried, obtain EGFR-Dextran-DOX antineoplastics.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (6)
1. a kind of antineoplastic, it is characterised in that including one kind in adriamycin and cinnamic acid, EGFR antibody and dextran
Nano-medicament carrier, the EGFR antibody are connected with the dextran nano-medicament carrier, in the adriamycin and cinnamic acid
One kind be connected to by hydrazone key on the dextran nano-medicament carrier, the dextran nano-medicament carrier includes carboxylic
The dextran nano-particle of base, the particle diameter of the dextran nano-particle of the carboxylated is 20~100 nanometers.
A kind of 2. antineoplastic according to claim 1, it is characterised in that matter in the adriamycin and cinnamic acid
It is 23% to measure percentage.
3. a kind of preparation method of antineoplastic, it is characterised in that comprise the following steps：
EGFR antibody and dextran nano-medicament carrier are mixed, and adds carbodiimide and n-hydroxysuccinimide obtains the
One mixed liquor, the pH value of first mixed liquor is adjusted to 4.5~5, reacts 2~4 hours at room temperature, isolate and purify to obtain
Intermediate product, the dextran nano-medicament carrier include the dextran nano-particle of carboxylated, the right side of the carboxylated
The particle diameter for revolving sugared acid anhydride nano-particle is 20~100 nanometers；
The intermediate product is dissolved in the solution containing intermediate product is obtained in solvent, the solution containing intermediate product is dripped
Add in hydrazine solution, and add carbodiimide and n-hydroxysuccinimide obtains the second mixed liquor, described second is mixed
Liquid isolated and purified to obtain amido modified intermediate product in room temperature reaction 20~25 hours；
The amido modified intermediate product is dissolved in solvent, one kind added in adriamycin and cinnamic acid, at 35~40 DEG C
Lower vibration 48~72 hours, obtains the antineoplastic after isolating and purifying.
4. the preparation method of antineoplastic according to claim 3, it is characterised in that the EGFR antibody and the right side
The mol ratio for revolving sugared acid anhydride nano-medicament carrier is 20~30:1.
5. the preparation method of antineoplastic according to claim 3, it is characterised in that it is described and add carbodiimide and
N-hydroxysuccinimide was obtained in the step of the first mixed liquor, the mol ratio of the carbodiimide and n-hydroxysuccinimide
For 5~10:1.
6. the preparation method of antineoplastic according to claim 3, it is characterised in that it is described and add carbodiimide and
N-hydroxysuccinimide was obtained in the step of the second mixed liquor, the mol ratio of the carbodiimide and n-hydroxysuccinimide
For 5~10:1.
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