Summary of the invention
Based on this, be necessary the graphene oxide pharmaceutical carrier that provides a kind of stability higher.
A kind of antitumor drug that comprises this graphene oxide pharmaceutical carrier and preparation method thereof further is provided.
A kind of stannic oxide/graphene nano pharmaceutical carrier comprises a plurality of nano-particle, and each nano-particle comprises the kernel that is formed by graphene oxide and the shell that is formed by pyrenyl disulfide group Polyethylene Glycol; Wherein, described pyrenyl disulfide group Polyethylene Glycol adheres to the surface of described graphene oxide by the π-pi-conjugated effect of pyrenyl and graphene oxide.
Among embodiment, the mass ratio of described graphene oxide and pyrenyl disulfide group Polyethylene Glycol is 1~2:10 therein.
Among embodiment, the particle diameter of described nano-particle is 70~100 nanometers therein.
A kind of antitumor drug comprises hydrophobic medicine and above-mentioned stannic oxide/graphene nano pharmaceutical carrier with antitumor curative effect, and described medicine is carried on the kernel of described stannic oxide/graphene nano pharmaceutical carrier by hydrophobic interaction.
Among embodiment, the mass ratio of described medicine and described stannic oxide/graphene nano pharmaceutical carrier is 1:11~12 therein.
Among embodiment, described medicine is amycin, paclitaxel or camptothecine therein.
A kind of preparing anti-tumor medicine method comprises the steps:
With graphene oxide, pyrenyl disulfide group Polyethylene Glycol and hydrophobic medicine with antitumor curative effect are added to the water and obtain mixture, with described mixture supersound process 2 hours under room temperature, filter, obtain antitumor drug after the lyophilizing, described antitumor drug comprises stannic oxide/graphene nano pharmaceutical carrier and hydrophobic medicine with antitumor curative effect, described stannic oxide/graphene nano pharmaceutical carrier comprises a plurality of nano-particle, and each nano-particle comprises the kernel that formed by graphene oxide and adheres to the shell that the surface of described kernel forms by pyrenyl disulfide group Polyethylene Glycol; Wherein, the surface that the π-pi-conjugated effect of described pyrenyl disulfide group Polyethylene Glycol by pyrenyl and graphene oxide adheres to described graphene oxide forms shell, and described hydrophobic medicine with antitumor curative effect is carried on the kernel of described stannic oxide/graphene nano pharmaceutical carrier by hydrophobic interaction.
Among embodiment, the mass ratio of described graphene oxide, pyrenyl disulfide group Polyethylene Glycol and medicine is 1~2:10:1 therein.
Among embodiment, in the described mixture, the concentration of described graphene oxide is 0.1~1mg/mL therein.
Among embodiment, described pyrenyl disulfide group Polyethylene Glycol prepares as follows therein:
By solid-to-liquid ratio 0.17g:88mg:30mL cystamine hydrochlorate and sodium hydroxide are added to the water and obtain first mixture, described first mixture was stirred under room temperature 30 minutes, revolve to steam in 45 ℃ of following vacuum again and dewater, add methylene chloride then and add in first mixture after described the dewatering, remove by filter precipitation, revolve in 30 ℃ of vacuum again and steam except dichloromethane, obtain cystamine;
Obtain second mixture by solid-to-liquid ratio 120mg:28mg:1g:30mL mixing 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, N-maloyl imines, carboxy methylation methoxy poly (ethylene glycol) and dichloromethane, with the reaction after 5 hours in nitrogen atmosphere, under the room temperature of described second mixture, in described reacted second mixture, splash into described cystamine, under room temperature, reacted 24 hours again, obtain Polyethylene Glycol and the amino chemical compound that is connected to form by disulfide bond;
By solid-to-liquid ratio 288mg:287mg:178mg:20mL pyrene butanoic acid, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, N-maloyl imines and dichloromethane are mixed and to be incorporated under the room temperature reaction 2 hours, add described Polyethylene Glycol and the amino chemical compound that is connected to form by disulfide bond then, reaction is 24 hours under room temperature, obtains described pyrenyl disulfide group Polyethylene Glycol.
Above-mentioned graphene oxide pharmaceutical carrier comprises a plurality of nano-particle, and each nano-particle comprises the kernel that is formed by graphene oxide and the shell that is formed by pyrenyl disulfide group Polyethylene Glycol.Wherein, pyrenyl disulfide group Polyethylene Glycol adheres to the surface formation hydrophilic outer shell of graphene oxide by the π-pi-conjugated effect of pyrenyl and graphene oxide, the modification of pyrenyl disulfide group Polyethylene Glycol makes this graphene oxide pharmaceutical carrier possess higher spatial stability, the characteristics of electrostatic stabilization, and stability is higher.
The specific embodiment
For above-mentioned purpose of the present invention, feature and advantage can be become apparent more, below in conjunction with accompanying drawing the specific embodiment of the present invention is described in detail.A lot of details have been set forth in the following description so that fully understand the present invention.But the present invention can implement much to be different from alternate manner described here, and those skilled in the art can do similar improvement under the situation of intension of the present invention, so the present invention is not subjected to the restriction of following public concrete enforcement.
The stannic oxide/graphene nano pharmaceutical carrier of one embodiment comprises a plurality of nano-particle, and each nano-particle comprises the kernel that formed by graphene oxide and adheres to the shell that the surface of kernel forms by pyrenyl disulfide group Polyethylene Glycol.This nano-particle in the form of sheets.
Pyrenyl disulfide group Polyethylene Glycol is by pyrene butanoic acid, cystamine hydrochlorate and carboxy methylation methoxy poly (ethylene glycol) (mPEG-COOH) reaction, form the pyrenyl disulfide group and pyrenyl linked to each other with disulfide group, the pyrenyl disulfide group is connected in the chemical compound that forms on the Polyethylene Glycol by disulfide group, is expressed as pyrene-S-S-PEG.
Pyrenyl disulfide group Polyethylene Glycol adheres to the surface formation hydrophilic outer shell of graphene oxide by the π-pi-conjugated effect of pyrenyl and graphene oxide.
Pyrenyl disulfide group Polyethylene Glycol is coated on inside with the graphene oxide of less stable, make this graphene oxide pharmaceutical carrier have the advantage that biocompatibility height, toxicity are low, drug loading is high of graphene oxide, the shortcoming of graphene oxide poor stability be can overcome again, biocompatibility height, stable high nano-medicament carrier obtained.This stannic oxide/graphene nano pharmaceutical carrier has higher spatial stability and electrostatic stabilization, and long circulating half-life.
Polyethylene Glycol (PEG) is positioned at the surface of nano-particle.The Polyethylene Glycol nano-particle has good biocompatibility, biodegradation is controlled and catabolite toxicity is low advantage, further makes this stannic oxide/graphene nano pharmaceutical carrier have higher biocompatibility.
Pyrene-S-S-PEG makes this stannic oxide/graphene nano pharmaceutical carrier can avoid immune identification with the formation hydrophilic outer shell that π-pi-conjugated effect adheres to the surface of graphene oxide, when using the carrier loaded medicine of this stannic oxide/graphene nano, can strengthen the half-life of drug system circulation, improve curative effect.
And graphene oxide forms the higher kernel of hydrophobicity, can pass through hydrophobic interaction load hydrophobic drug.
Preferably, the mass ratio of graphene oxide and pyrenyl disulfide group Polyethylene Glycol is 1~2:10, when guaranteeing that above-mentioned stannic oxide/graphene nano pharmaceutical carrier has advantages of higher stability, can give full play to the bigger advantage of graphene oxide drug loading and Polyethylene Glycol and be easy to the advantage that realizes that targeting control discharges.
Each nano-particle of above-mentioned stannic oxide/graphene nano pharmaceutical carrier is kernel with the graphene oxide, the pyrenyl of pyrene-S-S-PEG forms hydrophilic outer shell by the surface that π-pi-conjugated effect adheres to graphene oxide, containing under the aqueous environments of Reducing agent, this π-pi-conjugated effect disappears and the kernel of this stannic oxide/graphene nano pharmaceutical carrier is separated with shell, hydrophobic drug release.Above-mentioned stannic oxide/graphene nano pharmaceutical carrier has the reduction response, is easy to realize that the targeting of medicine discharges.
Preferably, be that kernel, pyrenyl disulfide group Polyethylene Glycol are that the particle diameter of the nano-particle of shell is 70~100 nanometers with the graphene oxide.
Fig. 1 is atomic force microscope (AFM) comparison diagram of graphene oxide and above-mentioned stannic oxide/graphene nano pharmaceutical carrier.Wherein, A is atomic force microscope (AFM) figure of graphene oxide, and B is the AFM figure of above-mentioned stannic oxide/graphene nano pharmaceutical carrier.As seen from Figure 1, above-mentioned stannic oxide/graphene nano pharmaceutical carrier is the same with graphene oxide, and particle diameter is little, and surface energy is big.
Fig. 2 is the lamella degree of contrast figure of graphene oxide and above-mentioned stannic oxide/graphene nano pharmaceutical carrier, and wherein, C represents graphene oxide, and D represents above-mentioned stannic oxide/graphene nano pharmaceutical carrier.As seen from Figure 2, the sheet layer height homogeneity of above-mentioned stannic oxide/graphene nano pharmaceutical carrier is better, and the stable higher of above-mentioned stannic oxide/graphene nano pharmaceutical carrier is described.
The antitumor drug of one embodiment comprises above-mentioned stannic oxide/graphene nano pharmaceutical carrier and hydrophobic medicine with antitumor curative effect.Hydrophobic medicine with antitumor curative effect is carried on the graphene oxide kernel of this stannic oxide/graphene nano pharmaceutical carrier by hydrophobic interaction, forms a plurality of antitumor drug nanoparticles.
Preferably, hydrophobic have the medicine of antitumor curative effect and the mass ratio of this graphene oxide is 1:11~12.
Hydrophobic medicine with antitumor curative effect is amycin, paclitaxel or camptothecine.
This antitumor drug is owing to adopt above-mentioned stannic oxide/graphene nano pharmaceutical carrier as carrier loaded hydrophobic medicine with antitumor curative effect, make this antitumor drug have higher spatial stability and electrostatic stabilization, be difficult for reuniting, can be brought into play curative effect by human body institute's metabolism well.Simultaneously this antitumor drug can be avoided immune identification, Xun Huan half-life in vivo, curative effect height.
And this antitumor drug has good biocompatibility, biodegradation is controlled and catabolite toxicity is low advantage.This antitumor drug also has the reduction response, is easy to realize that targeting control discharges.The release principle of this antitumor drug as shown in Figure 3, hydrophobic medicine with antitumor curative effect is example with the amycin, after the administration, this antitumor drug enters blood circulation, amycin does not have release substantially in blood circulation, antitumor drug enters Cytoplasm by endocytosis after arriving tumor locus, stimulate the π-pi-conjugated effect that makes pyrene-S-S-PEG shell and graphene oxide kernel to disappear by the reduction response then, and hydrophilic pyrene-S-S-PEG shell is dissolved in the Cytoplasm, thereby discharge hydrophobic medicine with antitumor curative effect, medicine is better discharged, the performance curative effect at the target spot position.
The preparing anti-tumor medicine method of one embodiment comprises the steps:
Graphene oxide, pyrenyl disulfide group Polyethylene Glycol and hydrophobic medicine with antitumor curative effect be added to the water obtain mixture, with this mixture supersound process 2 hours under room temperature, filter, obtain antitumor drug after the lyophilizing.
Wherein, antitumor drug comprises stannic oxide/graphene nano pharmaceutical carrier and hydrophobic medicine with antitumor curative effect.The stannic oxide/graphene nano pharmaceutical carrier comprises a plurality of nano-particle, and each nano-particle comprises the kernel that formed by graphene oxide and adheres to the shell that the surface of kernel forms by pyrenyl disulfide group Polyethylene Glycol.Wherein, pyrenyl disulfide group Polyethylene Glycol adheres to the surface formation shell of graphene oxide by the π-pi-conjugated effect of pyrenyl and graphene oxide.Hydrophobic medicine with antitumor curative effect is carried on the kernel of stannic oxide/graphene nano pharmaceutical carrier by hydrophobic interaction.
Hydrophobic medicine with antitumor curative effect is amycin, paclitaxel or camptothecine.
Preferably, graphene oxide, pyrenyl disulfide group Polyethylene Glycol and hydrophobic mass ratio with medicine of antitumor curative effect are 1~2:10:1.
When hydrophobic medicine with antitumor curative effect is amycin, because the water solublity of amycin is relatively poor, for the ease of preparation, with doxorubicin hydrochloride as the feedstock production antitumor drug, for in and doxorubicin hydrochloride in hydrochloric acid, in the mixture of graphene oxide, pyrenyl disulfide group Polyethylene Glycol, hydrophobic medicine with antitumor curative effect and water, add triethylamine, with in and hydrochloric acid in the doxorubicin hydrochloride.
The solid-to-liquid ratio of doxorubicin hydrochloride and triethylamine is 1mg:1.5 μ L.
Preferably, in the mixture of graphene oxide, pyrenyl disulfide group Polyethylene Glycol, hydrophobic medicine with antitumor curative effect and water, the concentration of graphene oxide is 0.1~1mg/mL, so that graphene oxide is scattered in the water preferably, avoid graphene oxide to reunite, with preparation good dispersion, antitumor drug that particle diameter is little.
Above-mentioned pyrenyl disulfide group Polyethylene Glycol is by pyrene butanoic acid, cystamine hydrochlorate and carboxy methylation methoxy poly (ethylene glycol) (mPEG-COOH) reaction, form the pyrenyl disulfide group and pyrenyl linked to each other with disulfide group, the pyrenyl disulfide group is connected in the chemical compound that forms on the Polyethylene Glycol by disulfide group.Pyrenyl disulfide group Polyethylene Glycol adopts following method preparation:
By solid-to-liquid ratio 0.17g:88mg:30mL cystamine hydrochlorate and sodium hydroxide are added to the water and obtain first mixture, first mixture was stirred under room temperature 30 minutes, revolve to steam in 45 ℃ of following vacuum again and dewater, in first mixture after the adding that adds methylene chloride then dewaters, remove by filter precipitation, revolve in 30 ℃ of vacuum again and steam except dichloromethane, obtain the weak yellow liquid cystamine.
Obtain second mixture by solid-to-liquid ratio 120mg:28mg:1g:30mL mixing 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl), N-maloyl imines (NHS), methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) and dichloromethane, with the reaction after 5 hours in nitrogen atmosphere, under the room temperature of second mixture, in reacted second mixture, splash into the cystamine for preparing, under room temperature, reacted 24 hours again, obtain Polyethylene Glycol and the amino chemical compound that is connected to form by disulfide bond, be expressed as mPEG-S-S-NH
2The mass ratio of cystamine and methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) is 0.15:1.
The carboxyl that EDCHCl and NHS are used for activation methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) (COOH), to improve the activity of carboxyl, improves reaction rate.
By solid-to-liquid ratio 288mg:287mg:178mg:20mL pyrene butanoic acid, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl), N-maloyl imines (NHS) and dichloromethane are mixed to be incorporated under the room temperature reaction 2 hours, add 0.5g Polyethylene Glycol and the amino chemical compound (mPEG-S-S-NH that is connected to form by disulfide bond then
2), reaction is 24 hours under room temperature, obtains pyrenyl disulfide group Polyethylene Glycol.
It is 5000 methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) that above-mentioned methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) is preferably weight average molecular weight.
The employing weight average molecular weight is 5000 methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) preparation pyrenyl disulfide group Polyethylene Glycol, with the pyrenyl disulfide group Polyethylene Glycol that obtains for the preparation of the stannic oxide/graphene nano pharmaceutical carrier, and when this carrier is used for the hydrophobic medicine with antitumor curative effect of load and forms antitumor drug, make this antitumor drug in blood circulation, be difficult to other organ picked-up, be beneficial to targeting and discharge, and the particle diameter of this antitumor drug is less.
Method is simple for above-mentioned preparing anti-tumor medicine, and the reaction condition gentleness is convenient to operation and is promoted.
It below is specific embodiment.
Embodiment 1
Take by weighing 0.17g cystamine hydrochlorate (1.1mmol) and 88mgNaOH(2.2mmol) add in the 30mL water and at room temperature stir 30min, revolve to steam in 45 ℃ of vacuum again and dewater.Dichloromethane adds in the said mixture, has filtered precipitation, and 30 ℃ of vacuum are revolved and steamed except dichloromethane again.Obtain the weak yellow liquid cystamine.Get 120mgEDCHCl (0.62mmol), 28mg NHS (0.23mmol), 1g mPEG-COOH(weight average molecular weight 5000) and 30mL dichloromethane N at room temperature
2Behind the reaction 5h, slowly splash into the 0.15g cystamine in the atmosphere, react 24h more at normal temperatures, obtain Polyethylene Glycol and the amino chemical compound (mPEG-S-S-NH that is connected to form by disulfide bond
2);
Pipette 288mg pyrene butanoic acid, 287mg EDCHCl, 178mgNHS at room temperature activate 2h in the 20mL dichloromethane after, add 0.5g mPEG-S-S-NH
2After, at room temperature react 24h after, obtain pyrenyl disulfide group Polyethylene Glycol;
Pipette the 1mg graphene oxide, 10mg pyrenyl disulfide group Polyethylene Glycol, 1mg doxorubicin hydrochloride (DXR), 1.5 μ L triethylamine, add 10mL water, under the room temperature, with the ultrasonic 2h of ultrasonic carbon-point, allow solvent evaporates during this time, filter then, obtain antitumor drug after the lyophilizing, this antitumor drug comprises stannic oxide/graphene nano pharmaceutical carrier and amycin, the stannic oxide/graphene nano pharmaceutical carrier comprises a plurality of nano-particle, each nano-particle comprises the kernel that formed by graphene oxide and adheres to the shell that the surface of stating kernel forms by pyrenyl disulfide group Polyethylene Glycol that amycin is carried on the kernel by hydrophobic interaction.
Above-mentioned antitumor drug is 5.5 at pH value, the release profiles I in the aqueous solution of the glutathion (GSH) of 0mM/L, be 5.5 at pH value, the release profiles II in the aqueous solution of the glutathion (GSH) of 10mM/L, be 7.4 at pH value, the release profiles III in the aqueous solution of the glutathion (GSH) of 0mM/L and be 7.4 at pH value, the release profiles IV in the aqueous solution of the glutathion (GSH) of 10mM/L as shown in Figure 4.As seen from Figure 4, be respectively 5.5 and at 7.4 o'clock at pH value, above-mentioned antitumor drug can discharge in the aqueous solution of the glutathion (GSH) of 10mM/L preferably, and As time goes on, the amount of the amycin of release increases gradually.
Embodiment 2
Take by weighing 0.17g cystamine hydrochlorate (1.1mmol) and 88mgNaOH(2.2mmol) add in the 30ml water and at room temperature stir 30min, revolve to steam in 45 ℃ of vacuum again and dewater.Dichloromethane adds in the said mixture, has filtered precipitation, and 30 ℃ of vacuum are revolved and steamed except dichloromethane again.Obtain the weak yellow liquid cystamine.Get 120mgEDCHCl (0.62mmol), 28mgNHS(0.23mmol), 1gmPEG-COOH(weight average molecular weight 5000), 30ml dichloromethane N at room temperature
2In the atmosphere behind the reaction 5h, slowly splash into after the 0.15g cystamine reacts 24h more at normal temperatures, obtain mPEG-S-S-NH
2
Pipette 288mg pyrene butanoic acid, 287mgEDCHCl, 178mgNHS at room temperature activate 2h in the 20ml dichloromethane after, add 0.5gmPEG-S-S-NH
2After, at room temperature react 24h after, obtain pyrene-S-S-PEG.
Pipette the 2mg graphene oxide, 10mg pyrene-S-S-PEG, 1mgDXR, 1.5 μ l triethylamine, add 10ml water, under the room temperature, with the ultrasonic 2h of ultrasonic carbon-point, allow solvent evaporates during this time, filter then, obtain antitumor drug after the lyophilizing, this antitumor drug comprises stannic oxide/graphene nano pharmaceutical carrier and amycin, the stannic oxide/graphene nano pharmaceutical carrier comprises a plurality of nano-particle, each nano-particle comprises the kernel that formed by graphene oxide and adheres to the shell that the surface of stating kernel forms by pyrenyl disulfide group Polyethylene Glycol that amycin is carried on the kernel by hydrophobic interaction.This antitumor drug is containing the GSH(gsh proglumide of 10mM/L) among the PBS of PH=5.5, its reduction stimuli responsive releasing effect is good.
Embodiment 3
Take by weighing 0.34g cystamine hydrochlorate (2.2mmol) and 176mgNaOH(4.4mmol) add in the 60mL water and at room temperature stir 30min, revolve to steam in 45 ℃ of vacuum again and dewater.Dichloromethane adds in the said mixture, has filtered precipitation, and 30 ℃ of vacuum are revolved and steamed except dichloromethane again.Obtain the weak yellow liquid cystamine.Get 240mgEDCHCl (1.24mmol), 56mg NHS (0.46mmol), 2g mPEG-COOH(weight average molecular weight 5000) and 60mL dichloromethane N at room temperature
2Behind the reaction 5h, slowly splash into the 0.30g cystamine in the atmosphere, react 24h more at normal temperatures, obtain Polyethylene Glycol and the amino chemical compound (mPEG-S-S-NH that is connected to form by disulfide bond
2);
Pipette 288mg pyrene butanoic acid, 287mg EDCHCl, 178mgNHS at room temperature activate 2h in the 20mL dichloromethane after, add 1.0g mPEG-S-S-NH
2After, at room temperature react 24h after, obtain pyrenyl disulfide group Polyethylene Glycol;
Pipette 1mg graphene oxide, 10mg pyrenyl disulfide group Polyethylene Glycol and 1mg paclitaxel and add 10mL water, under the room temperature, with the ultrasonic 2h of ultrasonic carbon-point, allow solvent evaporates during this time, obtain antitumor drug after filtration, the lyophilizing then, this antitumor drug comprises stannic oxide/graphene nano pharmaceutical carrier and paclitaxel, the stannic oxide/graphene nano pharmaceutical carrier comprises a plurality of nano-particle, each nano-particle comprises the kernel that formed by graphene oxide and adheres to the shell that the surface of stating kernel forms by pyrenyl disulfide group Polyethylene Glycol that paclitaxel is carried on the kernel by hydrophobic interaction.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.