CN104072765B - Modified polyethyleneimine and preparation method thereof, drug gene carrier system and preparation method thereof - Google Patents
Modified polyethyleneimine and preparation method thereof, drug gene carrier system and preparation method thereof Download PDFInfo
- Publication number
- CN104072765B CN104072765B CN201410326285.8A CN201410326285A CN104072765B CN 104072765 B CN104072765 B CN 104072765B CN 201410326285 A CN201410326285 A CN 201410326285A CN 104072765 B CN104072765 B CN 104072765B
- Authority
- CN
- China
- Prior art keywords
- present
- carrier system
- polyethyleneimine
- complex carrier
- drug gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- AFEDQQKVLBHDNO-UHFFFAOYSA-N CCC(CC)C(C)N Chemical compound CCC(CC)C(C)N AFEDQQKVLBHDNO-UHFFFAOYSA-N 0.000 description 1
- MIFWXJNZWLWCGL-UHFFFAOYSA-N CCCCNCCCN Chemical compound CCCCNCCCN MIFWXJNZWLWCGL-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention provides a kind of modified polyethyleneimine, is obtained by the reaction of N (β maleimidopropionic acids) hydrazides, sulfhydryl compound and polyethyleneimine.Using the modified polyethyleneimine of the invention provided as carrier loaded antineoplastic and gene, drug gene complex carrier system is obtained.Obtained drug gene complex carrier system can direct pulmonary administration, medicine and gene is rapidly achieved highly concentrated level in lung tumors tissue and reduce the distribution in other normal structures, realize dosed administration and toxic side effect is low.The invention provides a kind of preparation method of modified polyethyleneimine, a kind of preparation method of drug gene complex carrier system is also provided.
Description
Technical field
The invention belongs to bio-carrier field, more particularly to modified polyethyleneimine and preparation method thereof, drug gene are multiple
Close carrier system and preparation method thereof.
Background technology
Lung cancer is also known as primary bronchogenic carcinoma, is one of common malignant tumour.In recent years, the lung cancer morbidity rate of various countries and
The death rate steeply rises, and in some industrially developed country, such as Britain, the U.S., Canada, lung cancer has occupied deaths in men
It is the first.It is reported that the average 5 years survival rate of lung cancer is less than 10%, the life cycle of most of lung cancer patients is 2 years or so.
The method of current traditional treatment lung cancer has chemotherapy or radiation treatment, on the one hand, chemotherapy or radiation treatment exist side effect it is big,
The problems such as big, high recurrence rate is injured to body and multidrug resistance mechanism is produced；On the other hand, malignant tumour is to be related to polygenes
Relevant disease, single pair of some gene carries out function interference and is difficult to reach preferable therapeutic effect.
In recent years research is found, medicine and gene are carried altogether, builds the drug gene complex carrier system of highly effective and safe
System, cancer therapy drug and therapeutic gene are transported in tumour cell simultaneously, and gene therapy and chemotherapy can be made to play collaboration and made
With reaching ideal therapeutic effect.Medicine and multiple therapeutic genes can be total to by drug gene complex carrier system
Carry, ospc gene is related to while carrying out function interference to multiple；The multidrug resistance of cancer cell can be also effectively solved, cancer therapy drug is reduced
Toxic action to human normal cell, with huge development potentiality and application value.
When using drug gene complex carrier systematic treating lung cancer, the transmission of medicine and gene is that curative effect is controlled in influence
The one of the main reasons of fruit.In the prior art, drug gene complex carrier system means of conveyance are generally to be injected intravenously, but vein is noted
Penetrate toxicity big, medicine or gene is degradable, medicine or mrna concentration are easily reduced.
The content of the invention
It is an object of the invention to provide modified polyethyleneimine and preparation method thereof, drug gene complex carrier system and
Its preparation method.The drug gene complex carrier system that the modified polyethyleneimine provided using the present invention is prepared can be straight
Pulmonary administration is connect, medicine and gene is rapidly achieved highly concentrated level in lung tumors tissue and is reduced in other normal structures
Distribution, dosed administration can be realized and toxic side effect is low.
The present invention provides a kind of modified polyethyleneimine, by N- (β-maleimidopropionic acid) hydrazides, sulfhydryl compound
Obtained with polyethyleneimine reaction.
It is preferred that, the polyethyleneimine has structure shown in formula 1 or formula 2：
Wherein, a, b and c are the degree of polymerization, and the number-average molecular weight of the polyethyleneimine is 600~35000Da；
The sulfhydryl compound includes the one or more in TGA, mercaptopropionic acid and mercaptoethanol.
It is preferred that, the mol ratio of the polyethyleneimine and N- (β-maleimidopropionic acid) hydrazides is 1：(0.5~
100)。
The present invention provides a kind of preparation method of modified polyethyleneimine, comprises the following steps：
A) sulfhydryl compound and polyethyleneimine are reacted in a solvent, obtain sulfhydryl modified polyethyleneimine；
B) by N- (β-maleimidopropionic acid) hydrazides and the step A) obtained sulfhydryl modified polyethyleneimine mixes
Close, be modified reaction, obtain modified polyethyleneimine.
It is preferred that, the step A) in sulfhydryl compound and polyethyleneimine mol ratio be 1：(0.5~100)；
The step A) in polyethyleneimine and the step B) in N- (β-maleimidopropionic acid) hydrazides rub
You are than being 1：(0.5~100).
It is preferred that, the step A) in reaction temperature be 20~40 DEG C；
The step A) in reaction time be 12~48 hours.
The step B) in modified-reaction temperature be 20~40 DEG C；
The step B) in modified-reaction time be 0.5~2 hour.
The present invention provides a kind of drug gene complex carrier system, including antineoplastic, genetic stew and carrier；
Carrier preparation side described in the modified polyethyleneimine or above-mentioned technical proposal described in above-mentioned technical proposal
The modified polyethyleneimine that method is obtained.
It is preferred that, the mol ratio of the carrier and antineoplastic is 1：(0.5~100).
The present invention provides a kind of preparation method of drug gene complex carrier system, comprises the following steps：
1) antineoplastic, modified polyethyleneimine and solvent are mixed, is reacted, obtain medicament carrier system, institute
State modified polyethyleneimine any for the modified polyethyleneimine or claim 4~8 described in claims 1 to 3 any one
The modified polyethyleneimine that preparation method described in one is obtained；
2) by genetic stew and the step 1) mixing of obtained medicament carrier system, it is incubated, obtains drug gene
Complex carrier system.
It is preferred that, the step 1) in modified polyethyleneimine and antineoplastic mol ratio be 1：(0.5~100)；
The step 2) in genetic stew and medicament carrier system mass ratio be 1：(1~80).
The present invention provides a kind of modified polyethyleneimine, by N- (β-maleimidopropionic acid) hydrazides, sulfhydryl compound
Obtained with polyethyleneimine reaction.Carrier loaded antineoplastic and base are used as using the modified polyethyleneimine that the present invention is provided
Cause, obtains drug gene complex carrier system.The drug gene complex carrier system that the present invention is provided can direct pulmonary administration,
Medicine and gene is rapidly achieved highly concentrated level in lung tumors tissue and reduce the distribution in other normal structures, realize
Dosed administration and toxic side effect is low.Test result indicates that, the drug gene complex carrier system that the present invention is provided is with lung
The mode of administration is used for the treatment of lung's melanoma of mouse, and after treating 28 days, the weight of mouse lung is obviously reduced, and illustrates this
Inventing the drug gene complex carrier system provided being capable of directly pulmonary administration, and have good suppression effect to lung tumors
Really.
Embodiment
The invention provides a kind of modified polyethyleneimine, by N- (β-maleimidopropionic acid) hydrazides, sulfydryl chemical combination
Thing and polyethyleneimine reaction are obtained.
The modified polyethyleneimine that the present invention is provided can obtain medicine base as carrier loaded antineoplastic and gene
Because of complex carrier system, the drug gene complex carrier system that the present invention is provided being capable of directly pulmonary administration, and swollen to lung
Knurl has good inhibition.
In the present invention, the polyethyleneimine preferably has structure shown in formula 1 or formula 2：
Wherein, a, b and c are the degree of polymerization, and the number-average molecular weight of the polyethyleneimine is preferably 600~35000Da, more excellent
Elect 5000~30000Da, most preferably 10000~25000Da as.In the present invention, the sulfhydryl compound is preferably sulfydryl
One or more in acetic acid, mercaptopropionic acid and mercaptoethanol, more preferably TGA.In the present invention, the polyethylene
The mol ratio of imines and N- (β-maleimidopropionic acid) hydrazides is preferably 1：(0.5~100), more preferably 1：(10~
90), most preferably 1：(20~80).
The present invention does not have to the source of the polyethyleneimine, sulfhydryl compound and N- (β-maleimidopropionic acid) hydrazides
There is special limitation, the commercially available business of polyethyleneimine, sulfhydryl compound and N- (β-maleimidopropionic acid) hydrazides can be used
Product, also can prepare polyethyleneimine, sulfhydryl compound and N- (β-Maleimidos third according to well known to those skilled in the art
Acid) technical scheme of hydrazides voluntarily prepared.
The invention provides a kind of preparation method of modified polyethyleneimine, comprise the following steps：
A) sulfhydryl compound and polyethyleneimine are reacted in a solvent, sulfhydryl modified polyethyleneimine is obtained；
B) by N- (β-maleimidopropionic acid) hydrazides and the step A) obtained sulfhydryl modified polyethyleneimine mixes
Close, be modified reaction, obtain modified polyethyleneimine.
The present invention reacts sulfhydryl compound and polyethyleneimine in a solvent, obtains sulfhydryl modified polyethyleneimine.This
Invention mixes the sulfhydryl compound with activator preferably before sulfhydryl compound and polyethyleneimine react in a solvent,
Carry out priming reaction, the sulfhydryl compound activated.In the present invention, the species of the sulfhydryl compound and source with it is above-mentioned
The species of sulfhydryl compound described in technical scheme is consistent with source, will not be repeated here；The activator is preferably 1- second
Base -3- (3- dimethyl aminopropyls)-carbodiimides (EDC) and n-hydroxysuccinimide (NHS)；The sulfhydryl compound and
The mass ratio of activator is preferably 1：(5~15), more preferably 1：(8~13), most preferably 1：11；The temperature of the priming reaction
Degree is preferably 20~40 DEG C；More preferably 25~30 DEG C, without being heated or being cooled, directly reacted at room temperature i.e.
Can；The time of the priming reaction is preferably 2~12 hours, more preferably 4~8 hours, most preferably 6 hours.
After the activation for completing the sulfhydryl compound, the present invention exists the sulfhydryl compound and polyethyleneimine of the activation
Reacted in solvent, obtain sulfhydryl modified polyethyleneimine.The present invention is preferably by the sulfhydryl compound of activation, polyethyleneimine hydrochloric acid
Salt reacts in a solvent, obtains sulfhydryl modified polyethyleneimine.In the present invention, the polyethyleneimine amine hydrochlorate preferably according to
Following methods are obtained：
By polyethyleneimine and mixed in hydrochloric acid, polyethyleneimine acid solution is obtained；
The polyethyleneimine acid solution is freezed, polyethyleneimine amine hydrochlorate is obtained.
The present invention preferably mixes polyethyleneimine with water, polyethylenimine solution is obtained, by obtained polyethyleneimine
Solution obtains polyethyleneimine acid solution with mixed in hydrochloric acid.In the present invention, it is described that water used is mixed with polyethyleneimine
Preferably distilled water, more preferably secondary water, of the invention to the no spy of the consumption that water used is mixed with polyethyleneimine
Polyethyleneimine, can be completely dissolved by different limitation.In the present invention, the pH value of the polyethyleneimine acid solution
Preferably 6.8~8.0, more preferably 7.0~7.8, most preferably 7.2~7.5.The present invention is preferably by polyethyleneimine and hydrochloric acid
Solution is mixed, and obtains polyethyleneimine acid solution.The present invention does not have special limit to the consumption and concentration of the hydrochloric acid solution
System, can make the pH value of the polyethyleneimine acid solution within the above range.In order to in following technical proposals
Lyophilized to make a distinction, the present invention will be named as first and freeze to the lyophilized of polyethyleneimine acid solution.The present invention is to described the
One lyophilized method does not have special restriction, using lyophilized technical scheme well known to those skilled in the art.
In the present invention, the solvent that sulfhydryl compound and the polyethyleneimine reaction is used is preferably methanol and/or super
Pure water, more preferably methanol.In the present invention, the ultra-pure water can the production of specifically chosen millipore companies milli-Q
Water.The present invention does not have special limitation to the consumption of the solvent mixed with sulfhydryl compound and polyethyleneimine amine hydrochlorate,
The reaction environment of liquid can be provided for the mixing of the sulfhydryl compound and polyethyleneimine amine hydrochlorate.In the present invention
In, the mol ratio of the sulfhydryl compound and polyethyleneimine amine hydrochlorate is preferably 1：(0.5~100), more preferably 1：(1~
80), most preferably 1：(10~50).
To prevent sulfhydryl oxidase, the present invention preferably carries out the sulfhydryl compound and polyethyleneimine under protective gas atmosphere
The reaction of amine hydrochlorate, more preferably carries out the sulfhydryl compound and polyethyleneimine hydrochloric acid under closed protective gas atmosphere
The reaction of salt, the protective gas is preferably nitrogen or inert gas, more preferably nitrogen；The protective gas is passed through the time
Preferably 0.5~2 hour, more preferably 1 hour.In the present invention, it is described to sulfhydryl compound and polyethyleneimine amine hydrochlorate
The temperature of reaction is preferably 20~40 DEG C, more preferably 25~35 DEG C；In embodiments of the present invention, without being heated or being dropped
Temperature, the reaction of the sulfhydryl compound and polyethyleneimine amine hydrochlorate is directly carried out at room temperature；The time of the reaction
Preferably 12~48 hours, most preferably more preferably 20~40 hours, 24~36 hours.
Obtain after sulfhydryl modified polyethyleneimine, it is of the invention by N- (β-maleimidopropionic acid) hydrazides and the sulfydryl
Modified polyethyleneimine is mixed, and is modified reaction, is obtained modified polyethyleneimine.The present invention is preferably by N- (β-maleimide
Alanine) hydrazides and obtained sulfhydryl modified polyethyleneimine mix, be modified reaction, obtain being modified poly- second in a solvent
Alkene imines.In the present invention, it is described mixed with N- (β-maleimidopropionic acid) hydrazides and the sulfhydryl modified polyethyleneimine
The solvent of conjunction is preferably the one or more in ultra-pure water, dimethylformamide, dimethyl sulfoxide (DMSO) and methanol, more preferably ultrapure
Water and/or methanol, most preferably methanol.In the present invention, the ultra-pure water can specifically chosen millipore companies production
Milli-Q water.The present invention is to described and N- (β-maleimidopropionic acid) hydrazides and obtained sulfhydryl modified polyethyleneimine
The consumption of the solvent of mixing does not have special limitation, can be the N- (β-maleimidopropionic acid) hydrazides and obtained mercapto
The mixing of base modified polyethyleneimine provides liquid reactions environment；In the present invention, the N- (β-Maleimidos third
Acid) hydrazides source it is consistent with above-mentioned technical proposal, will not be repeated here.In the present invention, the polyethyleneimine with it is described
The mol ratio of N- (β-maleimidopropionic acid) hydrazides is preferably 1：(0.5~100), more preferably 1：(1~80), most preferably
For 1：(10~50).
In the present invention, the temperature of the modified-reaction is preferably 20~40 DEG C, more preferably 25~30 DEG C；In the present invention
Embodiment in, without being heated or being cooled, the modified-reaction directly at room temperature carry out；The present invention is used
Ellman methods determine sulfhydryl content, after sulfydryl reaction completely, you can stop reaction, the time of the modified-reaction is preferably 0.5
~2 hours, most preferably more preferably 0.8~1.5 hour, 0.9~1.3 hour.
Complete after the modified-reaction, the preferred solvent removed in the reaction solution that modified-reaction is obtained of the present invention is obtained
Modified polyethyleneimine.The present invention does not have special restriction to the method for the removal solvent, in the present invention, and the removal is molten
The method of agent is preferably revolving.The present invention is rotated without special limitation to described, using rotation well known to those skilled in the art
The technical scheme of steaming.
By in the reaction solution solvent remove after, the present invention preferably will remove solvent after modified polyethyleneimine with
Water is mixed, and is dialysed, obtains modified polyethyleneimine.In order to be made a distinction with the dialysis in following technical proposals, the present invention
Dialysis to modified polyethyleneimine is named as the first dialysis, it is in the present invention, described sub- with dried modified poly ethylene
Amine mixing water used is preferably distilled water, more preferably secondary water.The present invention is to described sub- with dried modified poly ethylene
The consumption of amine mixing water used does not have special limitation, can dissolve the dried modified polyethyleneimine.
First dialysis is carried out present invention preferably employs bag filter, the molecular cut off of the bag filter in first dialysis is preferably
1000~7000Da, more preferably 2000~6000Da, most preferably 3000~5000Da.
Complete after the first dialysis, the modified polyethyleneimine that the present invention preferably obtains the first dialysis carries out second and freezed,
Obtain modified polyethyleneimine.The present invention does not have special limitation to the described second lyophilized method, using people in the art
Lyophilized technical scheme known to member.
Present invention also offers a kind of drug gene complex carrier system, including antineoplastic, genetic stew and carrier,
The modification that carrier preparation method described in modified polyethyleneimine described in above-mentioned technical proposal or above-mentioned technical proposal is obtained
Polyethyleneimine.In the present invention, the drug gene complex carrier system can be expressed as medicine-gene vector system.
In the present invention, the antineoplastic preferably include one kind in adriamycin, daunorubicin and Epi-ADM or
It is several, more preferably including adriamycin and daunorubicin, most preferably including adriamycin；The genetic stew be preferably DNA and/
Or siRNA, more preferably siRNA.In the present invention, the mol ratio of the carrier and antineoplastic is preferably 1：(0.5~
100), more preferably 1：(1~80), most preferably 1：(10~50)；The mass ratio of the genetic stew and carrier is preferably 1：
(1~40), more preferably 1：(1~20), most preferably 1：(1~15).
Present invention also offers a kind of preparation method of drug gene complex carrier system, comprise the following steps：
1) antineoplastic, modified polyethyleneimine and solvent are mixed, is reacted, obtain medicament carrier system, institute
Modified polyethyleneimine preparation method described in modified polyethyleneimine described in above-mentioned technical proposal or above-mentioned technical proposal is stated to obtain
The modified polyethyleneimine arrived；
2) by genetic stew and the step 1) mixing of obtained medicament carrier system, it is incubated, obtains drug gene
Complex carrier system.
The present invention preferably mixes modified polyethyleneimine solution with antineoplastic drug solution, is reacted, obtains medicine
Carrier system.Specifically, in an embodiment of the present invention, can by the modified polyethyleneimine and antineoplastic respectively with
Solvent is mixed, and obtains modified polyethyleneimine solution and antineoplastic drug solution；Again by the modified polyethyleneimine solution and
Antineoplastic drug solution mixing is reacted, and obtains medicament carrier system.In the present invention, the species of the antineoplastic with
The species of antineoplastic is consistent in above-mentioned technical proposal, will not be repeated here；In the present invention, the modified polyethyleneimine
Mol ratio with antineoplastic is preferably 1：(0.5~100), more preferably 1：(1~80), most preferably 1：(10~50).
In the present invention, the pH value of the modified polyethyleneimine solution is preferably 6.8~8.0, and more preferably 7.0~7.6；With it is described
The solvent that modified polyethyleneimine or antineoplastic are mixed is preferably ultra-pure water, dimethylformamide, dimethyl sulfoxide (DMSO) and first
One or more in alcohol, more preferably ultra-pure water and/or methanol, most preferably methanol.In the present invention, the ultra-pure water can
The milli-Q water of specifically chosen millipore companies production.The present invention is to the solvent mixed with modified polyethyleneimine
Consumption does not have special limitation, and the modified polyethyleneimine solution can be diluted in above-mentioned pH value range；This hair
The bright consumption to the solvent mixed with antineoplastic does not have special limitation, can be completely molten by the antineoplastic
Solution.
In the present invention, the reaction of the antineoplastic and modified polyethyleneimine is carried out preferably under the conditions of lucifuge；
The time of the reaction of the antineoplastic and modified polyethyleneimine is preferably 12~72 hours, and more preferably 20~60 is small
When, most preferably 24~36 hours；The temperature of the reaction of the antineoplastic and modified polyethyleneimine is preferably 20~40
DEG C, more preferably 25~30 DEG C；In an embodiment of the present invention, without being heated or being cooled, the antineoplastic and change
Property polyethyleneimine reaction directly at room temperature carry out.
After the reaction for completing the modified polyethyleneimine and antineoplastic, the present invention is preferably by obtained reaction solution
Carry out second to dialyse, obtain medicament carrier system.Second dialysis is carried out present invention preferably employs bag filter, described second is saturating
The molecular cut off of bag filter in analysis is preferably 1000~7000Da, more preferably 2000~6000Da, and most preferably 3500
~4000Da；The time of second dialysis is preferably 24~72 hours, more preferably 30~60 hours, most preferably 36~48
Hour.The present invention changes water-dialyzing, the adjacent time for changing water-dialyzing twice during preferably being dialysed described second
It is preferably spaced 1~5 hour, more preferably 1.5~3 hours, most preferably 2 hours；The pH value of the water-dialyzing is preferably 6.8
~7.6, more preferably 7.0~7.4.
Complete after second dialysis, the medicament carrier system that the present invention preferably obtains dialysis carries out the 3rd and freezed, and obtains
To medicament carrier system.The present invention does not have special limitation to the described 3rd lyophilized method, ripe using those skilled in the art
The lyophilized technical scheme known.
Obtain after medicament carrier system, the present invention mixes genetic stew with the medicament carrier system, is incubated, obtained
To drug gene complex carrier system.The present invention preferably mixes genetic stew and medicament carrier system in sterilized water, carries out
It is incubated, obtains drug gene complex carrier system.Specifically, in an embodiment of the present invention, can be by genetic stew and medicine
Carrier system is dissolved in sterilized water respectively, obtains genetic stew aseptic aqueous solution and medicament carrier system aseptic aqueous solution；Again will
The genetic stew aseptic aqueous solution is mixed with medicament carrier system aseptic aqueous solution, is incubated, and is obtained drug gene and is combined
Carrier system.In order to improve composite effect, after medicament carrier system aseptic aqueous solution is obtained, the present invention is preferably by the medicine
Carrier system aseptic aqueous solution is filtered, to remove the bacterium in the medicament carrier system aseptic aqueous solution.The present invention is excellent
Choosing is filtered to medicament carrier system aseptic aqueous solution using miillpore filter, and the aperture of the miillpore filter is preferably 0.2~
0.3 μm, more preferably 0.22 μm.In the present invention, the species of the genetic stew and source and gene in above-mentioned technical proposal
The species of material is consistent with source, will not be repeated here.In the present invention, the quality of the medicament carrier system aseptic aqueous solution
Concentration is preferably 0.5~2mg/mL, more preferably 1mg/mL；The mass concentration of the genetic stew aseptic aqueous solution is preferably
0.01~0.1mg/mL, more preferably 0.02mg/mL；The present invention, without special limitation, can be adopted to the source of the sterilized water
, can also be according to the technical scheme well known to those skilled in the art for preparing sterilized water voluntarily with the commercial goods of the sterilized water
Prepare.Mixing and the consumption of the sterilized water of the present invention to the genetic stew and sterilized water do not have special limitation, can
The genetic stew and the medicament carrier system are completely dissolved.
In the present invention, the mass ratio of the genetic stew and the medicament carrier system is preferably 1：(1~80), it is more excellent
Elect 1 as：(2~70), most preferably 1：(10~50).In the present invention, the temperature of the incubation is preferably 0~35 DEG C, more excellent
Elect 10~30 DEG C, most preferably 20~25 DEG C as；In the present invention, it is described to be incubated directly in room without being heated or being cooled
Temperature is lower to be reacted；The time of the incubation is preferably 10~30min, more preferably 15~25min, is most preferably
20min。
The invention provides a kind of modified polyethyleneimine, prepared using the modified polyethyleneimine that the present invention is provided as carrier
Obtained drug gene complex carrier system can direct pulmonary administration, medicine and gene is quickly reached in lung tumors tissue
To the distribution of highly concentrated level and reduction in other normal structures, realize dosed administration and toxic side effect is low.
In order to further illustrate the present invention, the modified polyethyleneimine and its system provided with reference to embodiments the present invention
Preparation Method, drug gene complex carrier system and preparation method thereof are described in detail, but can not be understood as to the present invention
The restriction of protection domain.
Embodiment 1
200mg1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides and 100mg n-hydroxysuccinimides is molten
In 20mL methanol, 20 μ L TGAs are added thereto after dissolving completely, and activation 6 hours is carried out to TGA at 25 DEG C
Afterwards, the TGA activated；
PH value is adjusted using hydrochloric acid solution after 100mg molecular weight is dissolved for 1800Da polyethyleneimine with secondary water
It is lyophilized to obtain polyethyleneimine amine hydrochlorate for 7.0；
Obtained polyethyleneimine amine hydrochlorate is mixed with 10ml methanol, after dissolving completely, with the sulfydryl of obtained activation
Acetic acid is mixed, and leads to nitrogen after 1 hour, and reaction 24 hours of being held one's breath at 25 DEG C obtains sulfhydryl modified polyethyleneimine；
20mgN- (β-maleimidopropionic acid) hydrazides is mixed with 10ml methanol, gathered after dissolving completely with sulfhydryl modified
Aziridine is mixed, and sulfydryl response situation is monitored outside using Ellman methods, after being reacted 1.5 hours at 25 DEG C, stops reaction,
Obtained reaction solution is rotated, methanol is removed, then use molecule interception to be dialysed for 1000Da bag filter,
It is lyophilized, obtain modified polyethyleneimine.
20mg adriamycins are weighed, are mixed after adriamycin and modified polyethyleneimine are dissolved with methanol respectively, lucifuge reaction
After 24 hours, use molecule interception to be dialysed, freezed for 1000Da bag filter, obtain medicament carrier system；
10mg medicament carrier systems are taken, are mixed with sterilized water, the aseptic aqueous solution that concentration is 1mg/mL are configured to, using hole
Footpath is degerming for 0.22 μm of filtering with microporous membrane；Take 4mg plasmids pGL-3 to be mixed with sterilized water, be configured to the sterile of 0.02mg/mL
The aqueous solution；The aseptic aqueous solution of drug gene complex carrier system and plasmid pGL-3 aseptic aqueous solution are mixed, at 25 DEG C
30min is incubated, drug gene complex carrier system is obtained.
The present invention carries out cell toxicity test to obtained medicament carrier system, as a result shows, under condition of acidic pH, carefully
Cellular toxicity is 75%, and under the conditions of neutral and meta-alkalescence, cytotoxicity is 45%.Illustrate that the drug gene that the present invention is provided is answered
Carrier system is closed in the sour environment of tumour, with larger toxicity, and for the normal cellular environment of human body, and it is neutral and
Toxicity is smaller under the conditions of meta-alkalescence, and the injury to human normal cell is smaller.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 2
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 1.
Unlike, the consumption of adriamycin is 40mg in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 3
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 1.
Unlike, the consumption of adriamycin is 100mg in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 4
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 1.
Unlike, the molecular weight of polyethyleneimine is 600Da in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 5
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 2.
Unlike, the molecular weight of polyethyleneimine is 600Da in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 6
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 3.
Unlike, the molecular weight of polyethyleneimine is 600Da in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 7
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 1.
Unlike, the molecular weight of polyethyleneimine is 25000Da in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 8
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 2.
Unlike, the molecular weight of polyethyleneimine is 25000Da in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 9
Medicament carrier system and drug gene complex carrier system are obtained according to the technical scheme in the embodiment of the present invention 3.
Unlike, the molecular weight of polyethyleneimine is 25000Da in the present embodiment.
The present invention is measured the obtained particle diameter of medicament carrier system and surface potential, as a result as shown in table 1.Table
1 is the particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 is obtained.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
The particle diameter and surface potential for the medicament carrier system that the embodiment of the present invention 1~9 of table 1 is obtained
As can be seen from Table 1, the medicament carrier system particle diameter arrived of the embodiment of the present invention 1~9 is said all within 100nm
Bright medicament carrier system particle diameter provided in an embodiment of the present invention is easily absorbed by the body all in Nano grade；The embodiment of the present invention 1~
9 to medicament carrier system surface potential be on the occasion of, be conducive to and electronegative DNA molecular associate, formed medicine base
Because of complex carrier system.
Embodiment 10~18
Respectively drug gene complex carrier system is obtained according to the technical scheme in the embodiment of the present invention 1~9.Different
It is that the mass ratio of polyethyleneimine and plasmid is 2.5 in the embodiment of the present invention 10~18:1.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 19~27
Respectively drug gene complex carrier system is obtained according to the technical scheme in the embodiment of the present invention 1~9.Different
It is that the mass ratio of polyethyleneimine and plasmid is 5 in the embodiment of the present invention 19~27:1.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Embodiment 28~36
Respectively drug gene complex carrier system is obtained according to the technical scheme in the embodiment of the present invention 1~9.Different
It is that the mass ratio of polyethyleneimine and plasmid is 10 in the embodiment of the present invention 28~36:1.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 2.Table 2 is the particle diameter and surface electricity for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained
Position.
Comparative example 1
The polyethyleneimine for taking 100mg molecular weight to be 25000Da, is mixed with sterilized water, and it is 1mg/mL's to be configured to concentration
Aseptic aqueous solution, uses aperture degerming for 0.22 μm of filtering with microporous membrane；Take 2mg plasmids pGL-3 to be mixed with sterilized water, prepare
Into 0.02mg/mL aseptic aqueous solution；By the aseptic aqueous solution of drug gene complex carrier system and plasmid pGL-3 sterilized water
Solution is mixed, and is incubated 30min at 25 DEG C, is obtained gene vector system.
The present invention is measured the obtained particle diameter of gene vector system and surface potential, as a result as shown in table 2.Table
2 be the particle diameter and surface potential for the drug gene complex carrier system that the embodiment of the present invention 1~36 and comparative example 1 are obtained.
The particle diameter for the drug gene complex carrier system that the embodiment of the present invention 1~36 of table 2 and comparative example are obtained and surface electricity
Position
As can be seen from Table 2, the particle diameter for the drug gene complex carrier system that the embodiment of the present invention 1~36 is provided exists
Below 100nm, belongs to Nano grade, it is easy to be absorbed by the body.
Embodiment 37~72
Drug gene complex carrier system is obtained according to the technical scheme in the embodiment of the present invention 1~36.Unlike, this
The plasmid pGL-3 in embodiment 1~36 is replaced using Luc siRNA in inventive embodiments 37~72.
The present invention is measured the obtained particle diameter of drug gene complex carrier system and surface potential, as a result such as table
Shown in 3.Table 3 is the particle diameter and table for the drug gene complex carrier system that the embodiment of the present invention 37~72 and comparative example 2~3 are obtained
Face current potential.
Comparative example 2
Gene vector system is obtained according to the technical scheme in comparative example 1.Unlike, Luc is used in the present embodiment
SiRNA replaces the pGL-3 in comparative example 1.
The present invention is measured the obtained particle diameter of gene vector system and surface potential, as a result as shown in table 3.Table
3 be the particle diameter and surface potential for the drug gene complex carrier system that the embodiment of the present invention 37~72 and comparative example 2~3 are obtained.
Comparative example 3
Gene vector system is obtained according to the technical scheme in comparative example 1.Unlike, using negative right in the present embodiment
The pGL-3 in comparative example 1 is replaced according to Rev siRNA.
The present invention is measured the obtained particle diameter of gene vector system and surface potential, as a result as shown in table 3.Table
3 be the particle diameter and surface potential for the drug gene complex carrier system that the embodiment of the present invention 37~72 and comparative example 2~3 are obtained.
The particle diameter and table for the drug gene complex carrier system that the embodiment of the present invention 37~72 of table 3 and comparative example 2~3 are obtained
Face current potential
As can be seen from Table 3, the particle diameter for the drug gene complex carrier system that the embodiment of the present invention 1~36 is provided exists
Below 100nm, belongs to Nano grade, it is easy to be absorbed by the body.
Embodiment 73
HeLa cells are placed in the DMEM cell culture fluids containing 10% hyclone, are 37 DEG C, volume integral in temperature
Number is passed on every other day to be cultivated in 5% CO2gas incubator.
When cell fusion degree is 80~90%, using Trypsin Induced, count, with every hole 2 × 104Density plant in
In 96 well culture plates, overnight incubation.During transfection, the drug gene complex carrier system that embodiment 1 is obtained is added to 96 orifice plates
In, cultivate 48 hours.
After 48 hours, nutrient solution is discarded, after being washed twice with phosphate buffer, cell pyrolysis liquid is added in -80 DEG C of ice
Multigelation in case, adds luciferase substrate after piping and druming is uniform, uses spectrophotometric determination transfection efficiency.As a result such as the institute of table 4
Show, table 4 is the transfection efficiency in vitro for the drug gene complex carrier system that the embodiment of the present invention 73~108 and comparative example 4 are obtained.
Embodiment 74~108
The drug gene carrier system obtained according to the technical scheme in the embodiment of the present invention 73 to embodiment 2~36 is carried out
The test of transfection efficiency in vitro.As a result as shown in table 4, table 4 is the medicine that the embodiment of the present invention 73~108 and comparative example 4 are obtained
Gene complex carrier system transfection efficiency in vitro.
Comparative example 4
The gene vector system that technical scheme according to the embodiment of the present invention 73 is obtained to comparative example 1 carries out in-vitro transfection effect
The test of rate.As a result as shown in table 4, table 4 is the compound load of drug gene that the embodiment of the present invention 73~108 and comparative example 4 are obtained
System system transfection efficiency in vitro.
The drug gene complex carrier system transfection efficiency in vitro that the embodiment of the present invention 73~108 of table 4 and comparative example 4 are obtained
As can be seen from Table 4, the drug gene complex carrier system that the present invention is provided has suitable with polyethyleneimine
Transfection efficiency.
Embodiment 109~144
The drug gene obtained according to the technical scheme in the embodiment of the present invention 73 to the embodiment of the present invention 37~72 is combined
Carrier system carries out in-vitro transfection, and use can stablize the HeLa cells of expressing luciferase, transfect the plasmid of silence luciferase,
Calculate silence efficiency.As a result as shown in table 5, table 5 is the medicine base that the embodiment of the present invention 109~144 and comparative example 5~6 are obtained
Because complex carrier system gene suppresses efficiency.
Comparative example 5~6
The gene vector system obtained according to the technical scheme in the embodiment of the present invention 73 to comparative example 2~3 of the present invention enters
Row in-vitro transfection, use can stablize the HeLa cells of expressing luciferase, transfect the plasmid of silence luciferase, calculate silence effect
Rate.As a result as shown in table 5, table 5 is the drug gene complex carrier that the embodiment of the present invention 109~144 and comparative example 5~6 are obtained
System gene suppresses efficiency.
The drug gene complex carrier system gene silence that the embodiment of the present invention 109~144 of table 5 and comparative example 5~6 are obtained
Efficiency
| Embodiment | Transfection suppresses efficiency (%) | Embodiment | Gene silencing efficiency (%) |
| Comparative example 5 | 45 | 126 | 47 |
| Comparative example 6 | 9 | 127 | 41 |
| 109 | 39 | 128 | 45 |
| 110 | 49 | 129 | 58 |
| 111 | 36 | 130 | 45 |
| 112 | 38 | 131 | 51 |
| 113 | 45 | 132 | 40 |
| 114 | 45 | 133 | 52 |
| 115 | 57 | 134 | 52 |
| 116 | 46 | 135 | 42 |
| 117 | 43 | 136 | 42 |
| 118 | 41 | 137 | 38 |
| 119 | 50 | 138 | 39 |
| 120 | 40 | 139 | 36 |
| 121 | 50 | 140 | 49 |
| 122 | 46 | 141 | 38 |
| 123 | 36 | 142 | 53 |
| 124 | 40 | 143 | 50 |
| 125 | 46 | 144 | 52 |
As can be seen from Table 5, the gene for the drug gene complex carrier system that the present invention is provided suppresses efficiency with being used as sun
Property control comparative example 5 approach, illustrate the present invention provide drug gene complex carrier system gene suppression efficiency it is higher.
Embodiment 145
B16F10 cells are placed in the DMEM nutrient solutions containing 10% hyclone, are 5% in 37 DEG C, volume fraction
Cultivated in CO2gas incubator, fresh medium is changed every other day.
The C57 male mices that weight is 20g are selected, when cell fusion degree is 80~90%, with Trypsin Induced, are used
PBS is washed twice and counted, and passes through tail vein injection 1 × 104Cells/ only, starts lung's transfer melanoma occur on the 7th day.
The drug gene complex carrier system compounding pharmaceutical gene complex carrier system that the embodiment of the present invention 10 is obtained
The mass concentration of glucose solution, wherein gene is 0.1mg/mL, and the mass concentration of adriamycin is 0.2mg/mL.Opened from the 7th day
Begin, the glucose solution of obtained drug gene complex carrier system is injected directly into lung through pulmonary delivery device, carry out
Transfection in vivo.
After transfection in vivo 7 days, mouse euthanasia takes out lung, PBS is washed twice, addition lysate cracking, homogenate, plus
Enter luciferase substrate, use spectrophotometric determination transfection efficiency.As a result as shown in table 6, table 6 be the embodiment of the present invention 145~
153 and the obtained drug gene carrier system body of comparative example 7 in transfection efficiency.
Embodiment 146~153
The drug gene obtained according to the technical scheme in the embodiment of the present invention 145 to the embodiment of the present invention 11~18 is combined
Carrier system carries out the measure of internal transfection efficiency, as a result as shown in table 6.Table 6 is the embodiment of the present invention 145~153 and compared
Transfection efficiency in the drug gene carrier system body that example 7 is obtained.
Comparative example 7
The gene vector system obtained according to the technical scheme in the embodiment of the present invention 145 to comparative example 1 of the present invention is carried out
The measure of internal transfection efficiency, as a result as shown in table 6.Table 6 is the medicine that the embodiment of the present invention 145~153 and comparative example 7 are obtained
Transfection efficiency in gene vector system body.
Transfection efficiency in the drug gene carrier system body that the embodiment of the present invention 145~153 of table 6 and comparative example 7 are obtained
| Embodiment | Internal transfection efficiency (RLU/mgProtein) |
| Comparative example 7 | 1.8×107 |
| 145 | 3.4×106 |
| 146 | 1.3×107 |
| 147 | 4.7×107 |
| 148 | 1.8×107 |
| 149 | 2.3×107 |
| 150 | 3.1×107 |
| 151 | 1.4×107 |
| 152 | 2.9×107 |
| 153 | 7.8×107 |
Embodiment 154
B16F10 cells are placed in the DMEM nutrient solutions containing 10% hyclone, are 5% in 37 DEG C, volume fraction
Cultivated in CO2gas incubator, fresh medium is changed every other day.
The C57 male mices that weight is 18g are selected, when cell fusion degree is 80~90%, with Trypsin Induced, are used
PBS is washed twice and counted, and passes through tail vein injection 1 × 104Cells/ only, starts lung's transfer melanoma occur on the 7th day.
The drug gene complex carrier system compounding pharmaceutical gene complex carrier system that the embodiment of the present invention 46 is obtained
Glucose solution, wherein gene Survivin siRNA mass concentration are 0.1mg/mL.Since the 7th day, by obtained medicine
The glucose solution of thing gene complex carrier system injects directly to lung through pulmonary delivery device, is transfected in vivo, weekly
Injection is once.
After treatment the 28th day, mouse is euthanized, lung is taken out and weighs.As a result as shown in table 7, table 7 is the present invention
The drug gene complex carrier system in-vivo tumour histamine result that embodiment 154~162 and comparative example 8~12 are obtained.
Embodiment 155~162
The drug gene obtained according to the technical scheme in the embodiment of the present invention 154 to the embodiment of the present invention 47~54 is combined
Carrier system suppress in vivo the test of tumour, as a result as shown in table 7, and table 7 is the embodiment of the present invention 154~162 and compared
The drug gene complex carrier system in-vivo tumour histamine result that example 8~12 is obtained.
Comparative example 8
Select the C57 male mices that weight is 18g；
The drug gene complex carrier system compounding pharmaceutical gene complex carrier system that the embodiment of the present invention 46 is obtained
Glucose solution, wherein gene Survivin siRNA mass concentration are 0.1mg/mL；
The glucose solution of obtained drug gene complex carrier system is injected directly into lung through pulmonary delivery device,
Transfected, sprayed weekly once in vivo.
After treatment the 28th day, mouse is euthanized, lung is taken out and weighs.As a result as shown in table 7, table 7 is the present invention
The drug gene complex carrier system in-vivo tumour histamine result that embodiment 154~162 and comparative example 8~12 are obtained.
Comparative example 9
The test for according to the technical scheme in the embodiment of the present invention 154 transfect in Mice Body, unlike, this implementation
Example replaces the drug gene complex carrier system in embodiment 154 using 5% glucose solution.Test result is as shown in table 7,
Table 7 is that the drug gene complex carrier system in-vivo tumour that the embodiment of the present invention 154~162 and comparative example 8~12 are obtained suppresses
As a result.
Comparative example 10
The test for according to the technical scheme in the embodiment of the present invention 154 transfect in Mice Body, unlike, this implementation
Example replaces the drug gene complex carrier system in embodiment 154 using Survivin siRNA.Test result is as shown in table 7,
Table 7 is that the drug gene complex carrier system in-vivo tumour that the embodiment of the present invention 154~162 and comparative example 8~12 are obtained suppresses
As a result.
Comparative example 11~12
The gene vector system obtained according to the technical scheme in the embodiment of the present invention 154 to comparative example 2~3 of the present invention enters
The test of transfection in row Mice Body, as shown in table 7, table 7 is the embodiment of the present invention 154~162 and comparative example 8~12 to test result
Obtained drug gene complex carrier system in-vivo tumour histamine result.
Swollen in the drug gene complex carrier system body that the embodiment of the present invention 154~162 of table 7 and comparative example 8~12 are obtained
Knurl histamine result
| Embodiment | The weight (mg) of lung |
| Comparative example 8 | 212 |
| Comparative example 9 | 761 |
| Comparative example 10 | 742 |
| Comparative example 11 | 743 |
| Comparative example 12 | 751 |
| 154 | 305 |
| 155 | 253 |
| 156 | 312 |
| 157 | 285 |
| 158 | 234 |
| 159 | 267 |
| 160 | 314 |
| 161 | 241 |
| 162 | 264 |
Because B16F10 is high transfer murine melanoma, therefore lung's weight is to weigh the important indicator of therapeutic effect.
As can be seen from Table 7, the mouse lung weight of the drug gene complex carrier system of the invention provided has been used close to comparative example 8
In do not plant the mouse lung weight of knurl, illustrate that the drug gene complex carrier system that the present invention is provided is applied to pulmonary administration, and
And there is good inhibition to tumour.
Embodiment 163
The C57 male mices that 7 weight of selection are 18g, are anaesthetized mouse using intraperitoneal injection, using pulmonary delivery device
The drug gene complex carrier system that the embodiment of the present invention 46 is obtained is sprayed to lung, wherein, the consumption of adriamycin is every
Mouse 20 μ g, siRNA consumption are the siRNA of the every μ g Cy5.5 of mouse 20 mark.Respectively 0.5, take within 1,2,3,4,5,7 days
Mouse is put to death, and lung is in vitro, and imaging is checked mouse lung deposition conditions, as a result shown, at the 7th day, also some drugs and base
Because being deposited on mouse lung.
Embodiment 164~171
The drug gene that the embodiment of the present invention 47~54 is obtained is combined according to the technical scheme in the embodiment of the present invention 163
Carrier system is sprayed to lung, checks mouse lung deposition conditions, at the 7th day, also some drugs and gene are deposited on mouse
Lung.As a result show, the drug gene complex carrier system for being obtained the embodiment of the present invention 46~54 using pulmonary administration mode
Lung is injected directly to, not only drug loss is few, and it is long in the pulmonary deposition time, there are some drugs and gene heavy at the 7th day
Product is in lung.Further demonstrate that, the drug gene complex carrier system that provides of the present invention is suitable for pulmonary administration, and present compared with
Good effect.
Comparative example 13~14
Free medicine and gene are injected directly into lung according to the technical scheme in the embodiment of the present invention 163, checked
Mouse lung deposition conditions, as a result show, at the 7th day, mouse lung is not almost deposited.
Embodiment 172
The drug gene complex carrier for obtaining the embodiment of the present invention 46 according to the technical scheme in the embodiment of the present invention 163
System is sprayed to lung, checks mouse lung deposition conditions.Unlike, the present embodiment selects the C57 that 9 weight are 20g altogether
Male mice, respectively 0.5, after 1,2,4,6,8,12,24,48 hours, mouse is put to death, lung is in vitro, mouse is checked in imaging
Pulmonary deposition situation, as a result shows, in 48 hours, and mouse lung deposition change is little, illustrates what the embodiment of the present invention 172 was obtained
Drug gene complex carrier system being capable of directly pulmonary administration, and drug loss amount is few.
Embodiment 173~180
The drug gene that the embodiment of the present invention 47~54 is obtained is combined according to the technical scheme in the embodiment of the present invention 172
Carrier system is sprayed to lung, is checked mouse lung deposition conditions, is as a result shown, in 48 hours, and the change of mouse lung deposition is not
Greatly, illustrate that the drug gene complex carrier system that the embodiment of the present invention 173~180 is obtained being capable of directly pulmonary administration, and medicine
Thing loss amount is few.
Comparative example 15~16
Free medicine and gene are injected directly into lung according to the technical scheme in the embodiment of the present invention 172, checked
Mouse lung deposition conditions, as a result show, in 48 hours, mouse lung deposition change greatly, after 48 hours, drug loss amount compared with
Greatly.
Comparative example 17~25
The drug gene obtained according to the technical scheme in the embodiment of the present invention 172 to the embodiment of the present invention 46~54 is combined
Carrier system carries out pulmonary deposition test, checks mouse lung deposition conditions.Unlike, adopted in comparative example 17~25 of the present invention
The pulmonary administration in embodiment 172 is replaced with the mode of intravenous injection.
Comparative example 26~27
The gene vector system obtained according to the technical scheme in the embodiment of the present invention 172 to comparative example 2~3 of the present invention enters
Row pulmonary deposition is tested, and checks mouse lung deposition conditions.Unlike, using intravenous injection in comparative example 26~27 of the present invention
Mode replace embodiment 172 in pulmonary administration.
Shown by the experimental data of the embodiment of the present invention 172~180 and comparative example 15~27, the medicine base that the present invention is provided
Because complex carrier system can direct pulmonary administration, compared with tail vein is administered, drug gene is answered using pulmonary administration methods
Close carrier system and be directly injected to lung, drug loss amount is few, many and the residence time is long in pulmonary deposition.
The invention provides a kind of modified polyethyleneimine, obtained using the modified polyethyleneimine that the present invention is provided as carrier
Drug gene complex carrier system can direct pulmonary administration, test result indicates that, the drug gene that the present invention is provided is answered
Close carrier system is used for the treatment of lung's melanoma of mouse in the way of pulmonary administration, after treating 28 days, the lung of mouse
Weight is obviously reduced, and illustrates that the drug gene complex carrier system that the present invention is provided can not only directly pulmonary administration, and right
Tumour has good inhibition.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (4)
1. a kind of drug gene complex carrier system, including antineoplastic, genetic stew and carrier；
The carrier is modified polyethyleneimine；
The modified polyethyleneimine is reacted by N- (β-maleimidopropionic acid) hydrazides, sulfhydryl compound and polyethyleneimine
Obtain；
The polyethyleneimine has structure shown in formula 1 or formula 2：
Wherein, a, b and c are the degree of polymerization, and the number-average molecular weight of the polyethyleneimine is 600~25000Da；
One or more of the sulfhydryl compound in TGA, mercaptopropionic acid and mercaptoethanol；
The mass ratio of the carrier and antineoplastic is 5:1、5:2 or 1:1.
2. drug gene complex carrier system according to claim 1, it is characterised in that the polyethyleneimine and N-
The mol ratio of (β-maleimidopropionic acid) hydrazides is 1：(0.5~100).
3. a kind of preparation method of drug gene complex carrier system, comprises the following steps：
1) antineoplastic, modified polyethyleneimine and solvent are mixed, is reacted, obtain medicament carrier system, it is described to change
Property polyethyleneimine by N- (β-maleimidopropionic acid) hydrazides, sulfhydryl compound and polyethyleneimine reaction obtain；
The polyethyleneimine has structure shown in formula 1 or formula 2：
Wherein, a, b and c are the degree of polymerization, and the number-average molecular weight of the polyethyleneimine is 600~25000Da；
One or more of the sulfhydryl compound in TGA, mercaptopropionic acid and mercaptoethanol；
The mass ratio of the modified polyethyleneimine and antineoplastic is 5:1、5:2 or 1:1；
2) by genetic stew and the step 1) mixing of obtained medicament carrier system, it is incubated, obtains drug gene and be combined
Carrier system.
4. preparation method according to claim 3, it is characterised in that the step 2) in genetic stew and pharmaceutical carrier system
The mass ratio of system is 1：(1~80).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410326285.8A CN104072765B (en) | 2014-07-09 | 2014-07-09 | Modified polyethyleneimine and preparation method thereof, drug gene carrier system and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410326285.8A CN104072765B (en) | 2014-07-09 | 2014-07-09 | Modified polyethyleneimine and preparation method thereof, drug gene carrier system and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104072765A CN104072765A (en) | 2014-10-01 |
| CN104072765B true CN104072765B (en) | 2017-07-28 |
Family
ID=51594394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410326285.8A Active CN104072765B (en) | 2014-07-09 | 2014-07-09 | Modified polyethyleneimine and preparation method thereof, drug gene carrier system and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104072765B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104800861A (en) * | 2015-05-06 | 2015-07-29 | 中国科学院长春应用化学研究所 | Drug-gene loading system and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1761489A (en) * | 2003-02-17 | 2006-04-19 | 艾波顿有限公司 | Conjugates for medical imaging comprising carrier, targeting moiety and a contrast agent |
| CN101265477A (en) * | 2008-04-25 | 2008-09-17 | 浙江大学 | Method for preparing glutathione response shell disulfide bond crosslinking non-virogene vector |
| CN101711735A (en) * | 2009-12-10 | 2010-05-26 | 于美丽 | Slow-release medicament release system as well as preparation method and application thereof |
| CN103030813A (en) * | 2012-12-21 | 2013-04-10 | 深圳先进技术研究院 | Preparation method of chitosan grafted polyethyleneimine non-viral transgene vector |
| WO2014053521A2 (en) * | 2012-10-02 | 2014-04-10 | Novartis Ag | Nonlinear saccharide conjugates |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100766820B1 (en) * | 2006-01-23 | 2007-10-17 | 광주과학기술원 | Transmucosal Delivery System for Proteins or Peptides |
| JP2009508852A (en) * | 2006-01-23 | 2009-03-05 | クワンジュ インスティチュート オブ サイエンス アンド テクノロジー | Conjugate in which pharmacologically active substance and mucoadhesive polymer are covalently bonded, and method for transmucosal delivery of pharmacologically active substance using the same |
| CN101085356A (en) * | 2007-06-21 | 2007-12-12 | 中国科学院长春应用化学研究所 | Non-virogene transfection carrier, complex particles of the same and plasmid DNA, preparing method and using method |
| CN103497961B (en) * | 2013-09-25 | 2016-07-06 | 中国科学院长春应用化学研究所 | A kind of gene vector system and preparation method thereof |
-
2014
- 2014-07-09 CN CN201410326285.8A patent/CN104072765B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1761489A (en) * | 2003-02-17 | 2006-04-19 | 艾波顿有限公司 | Conjugates for medical imaging comprising carrier, targeting moiety and a contrast agent |
| CN101265477A (en) * | 2008-04-25 | 2008-09-17 | 浙江大学 | Method for preparing glutathione response shell disulfide bond crosslinking non-virogene vector |
| CN101711735A (en) * | 2009-12-10 | 2010-05-26 | 于美丽 | Slow-release medicament release system as well as preparation method and application thereof |
| WO2014053521A2 (en) * | 2012-10-02 | 2014-04-10 | Novartis Ag | Nonlinear saccharide conjugates |
| CN103030813A (en) * | 2012-12-21 | 2013-04-10 | 深圳先进技术研究院 | Preparation method of chitosan grafted polyethyleneimine non-viral transgene vector |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104072765A (en) | 2014-10-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Tumor hypoxia-activated combinatorial nanomedicine triggers systemic antitumor immunity to effectively eradicate advanced breast cancer | |
| Ye et al. | An integrated therapeutic delivery system for enhanced treatment of hepatocellular carcinoma | |
| CN100536924C (en) | Method for preparing drug administration carrier of gene with polyethylene imine beautify chitosan | |
| CN103099782B (en) | Core-shell type nano medical granule, preparation method and application thereof | |
| CN102816795B (en) | Genetic carrier system and preparation method thereof | |
| CN103768080A (en) | Targeting preparation for resisting drug-resistant tumor, as well as preparation method and application thereof | |
| AU2022211878B9 (en) | Targeting Degradable Nano-Drug Carrier for Chemo/Photothermal Synergistic Therapy and Preparation Method Thereof | |
| Xiang et al. | Endogenous Fe2+-activated ROS nanoamplifier for esterase-responsive and photoacoustic imaging-monitored therapeutic improvement | |
| CN108042490A (en) | Nano medicament carrying system, its preparation method, pharmaceutical composition and the application in treating cancer | |
| Yang et al. | BSA stabilized photothermal-fenton reactor with cisplatin for chemo/chemodynamic cascade oncotherapy | |
| CN107296962A (en) | Chemical drug/gene cotransports the preparation method and application of functionalized carbon nano-tube | |
| CN108384810A (en) | A kind of cationic gene carriers and preparation method thereof of high transfection efficiency low cytotoxicity | |
| WO2015188423A1 (en) | Guanidine hypoglycemic drug-polysaccharide conjugate, preparation method therefor and use thereof | |
| Zhong et al. | Polyhedral oligomeric silsesquioxane‐based nanoparticles for efficient chemotherapy of glioblastoma | |
| CN104072765B (en) | Modified polyethyleneimine and preparation method thereof, drug gene carrier system and preparation method thereof | |
| Cong et al. | How to exploit different endocytosis pathways to allow selective delivery of anticancer drugs to cancer cells over healthy cells | |
| CN104784700B (en) | A kind of medicine carries the preparation method of compound, micella and micella altogether | |
| Wang et al. | Functionalized biological metal–organic framework with nanosized coronal structure and hierarchical wrapping pattern for enhanced targeting therapy | |
| CN104072766B (en) | A kind of for carrying medicament with the carrier of gene, medicine-gene vector system and preparation method thereof | |
| CN106729746A (en) | To FAP α enzymes, the preparation method and applications of the tumor infiltrating nanosystems of the particle diameter shrinkage type of reducing environment sensitive | |
| CN105734075A (en) | Carrier for interfering with expression of gene ABCB5 and application of carrier in tumor stem cell treatment | |
| CN107899018B (en) | CD44 targeted chondroitin sulfate-adriamycin conjugate and PLGA mixed micelle thereof | |
| CN108969479B (en) | Method for constructing reduction response type anticancer nano-drug by polypeptide-drug co-assembly | |
| CN114652699B (en) | Size-transition type nano drug delivery carrier and preparation method and application thereof | |
| CN103834035B (en) | A kind of cationization laminarin and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |