CN104817688B - A kind of reversible nanogel of surface charge and preparation method thereof and a kind of reversible nanogel medicine carrying granule of surface charge - Google Patents
A kind of reversible nanogel of surface charge and preparation method thereof and a kind of reversible nanogel medicine carrying granule of surface charge Download PDFInfo
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Abstract
The preparation method that the invention provides the reversible nanogel of a kind of surface charge, comprise the following steps: A) initiator and the Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride with morpholine group is dissolved in organic solvent and reacts, obtain mixed solution；B) there is the compound of formula (I) structure, the compound with formula (II) structure and step A) mixed solution that obtains mixes, reacts, obtain reactant liquor；C) by step B) reactant liquor that obtains mixes with organic solvent, filters, obtain the reversible nanogel of surface charge；Adopt the reversible nanogel medicine carrying granule of surface charge provided by the invention to human non-toxic's side effect, and there is good water solublity, stability and biocompatibility.
Description
Technical field
The invention belongs to polymer drug carrier technique field, be specifically related to a kind of reversible nanogel of surface charge and preparation method thereof and a kind of reversible nanogel medicine carrying granule of surface charge.
Background technology
Tumor has become as one of most serious disease threatening human health.Anti-cancer therapies conventional clinically has operative treatment, radiotherapy and chemotherapy etc..
Wherein, operative treatment is the first-selected Therapeutic Method of early-stage cancer.Operative treatment cancer is cancerous tissue to be carried out all or the excision of local, and action effect is directly rapid.But operation cannot be accomplished thoroughly to remove cancerous cell, it is impossible to eliminate minimal disease, and the cancer patient for having occurred and that transfer is only capable of doing palliative local excision.Further, since the damage that operation brings to body, the immunity of patient can be made to reduce, the postoperative a series of complication of easy appearance.
Radiotherapy is by the roentgenization tumor of various different-energies, to suppress and to kill cancerous cell.It makes the ribonucleic acid long-chain in cancerous cell core suffer fatefulue destruction mainly by lonizing radiation, finally causes it dead.But, radiotherapy can not kill all of cancerous cell, and can reduce body immunity, transferred for cancer, diffusion patient, be merely able to play the effect of palliative therapy.
Therefore, chemotherapy is the therapy approach that oncotherapy is the most frequently used.Chemotherapy is to utilize chemicals to stop the propagation of cancerous cell, infiltration, transfer, until finally killing a kind of therapeutic modality of cancerous cell.Quickly being distributed to whole body after cancer therapy drug entrance is internal, the tumor that both can kill local also can kill the tumor of metastasis.Have whole body to send out tumor and the Advanced cancers of tendency for some, chemotherapy be main, be also unique selectable Therapeutic Method.But the antitumor drug used by chemotherapy is while killing cancerous cell clinically, also normal tissue cell non-selectivity kills, therefore, poisonous side effect of medicine is big, and, the defects such as antitumor drug exists water solublity and poor stability in application process, poor biocompatibility, thus limiting tumour medicine application in treatment cancer.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is in that to provide a kind of reversible nanogel of surface charge and preparation method thereof and a kind of reversible nanogel medicine carrying granule of surface charge.Adopt the reversible nanogel medicine carrying granule of surface charge provided by the invention to human non-toxic's side effect, and there is good water solublity, stability and biocompatibility.
The preparation method that the invention provides the reversible nanogel of a kind of surface charge, comprises the following steps:
A) initiator and the Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride with morpholine group is dissolved in organic solvent and reacts, obtain mixed solution；
B) there is the compound of formula (I) structure, the compound with formula (II) structure and step A) mixed solution that obtains mixes, reacts, obtain reactant liquor；
C) by step B) reactant liquor that obtains mixes with organic solvent, filters, obtain the reversible nanogel of surface charge；
Preferably, the described initiator with morpholine group one in formula (101)～(104)
Preferably, described Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride has formula (III) structure,
Wherein n is the degree of polymerization, 1≤n≤5.
Preferably, step A) described in organic solvent selected from N ' dinethylformamide, dioxane or chloroform, step B) described in organic solvent be ether.
Preferably, the described initiator with morpholine group is 1:(5～50 with the mol ratio of Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride)；Described is 1:(2～20 with the initiator of morpholine group with the mol ratio of the compound with formula (I) structure)；Described is 1:(2～20 with the initiator of morpholine group with the mol ratio of the compound with formula (II) structure).
Preferably, step A) temperature of described reaction is 15～50 DEG C, the time of described reaction is 48 hours～168 hours.
Preferably, step B) temperature of described reaction is 15～50 DEG C, the time of described reaction is 48 hours～168 hours.
Present invention also offers the reversible nanogel of surface charge that the preparation method described in a kind of the claims prepares.
Present invention also offers a kind of reversible nanogel medicine carrying granule of surface charge, antitumor drug and the preparation method described in the claims the reversible nanogel of surface charge prepared forms.
Preferably, described antitumor drug is selected from amycin, epirubicin, Perarubicin, paclitaxel, Docetaxel, cisplatin, carboplatin, oxaliplatin, bortezomib, camptothecine or shikonin.
Compared with prior art, the preparation method that the invention provides the reversible nanogel of a kind of surface charge, comprise the following steps: A) initiator and the Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride with morpholine group is dissolved in organic solvent and reacts, obtain mixed solution；B) there is the compound of formula (I) structure, the compound with formula (II) structure and step A) mixed solution that obtains mixes, reacts, obtain reactant liquor；C) by step B) reactant liquor that obtains mixes with organic solvent, filters, obtain the reversible nanogel of surface charge；
Nanogel medicine carrying particle surface provided by the invention, with morpholine group, is electric neutrality when the pH of blood, and is electropositive at tumor tissues, thus extending blood circulation time and making described nanoparticle easily by tumor tissues endocytosis.And, this nanogel biocatalytic particle forms nanogel structure by disulfide bond crosslinking, nanogel medicine carrying granule is obtained after medicine carrying, described medicine carrying granule is in the enrichment of tumor locus targeting and by tumor cell endocytosis, can fast fracture under disulfide bond homoglutathion concentration in tumor cell, realize the medicine intelligence release of inside tumor cells, reach to suppress the effect of tumor.Additionally, this nanogel medicine carrying granule has the shell of oligomeric ethylene glycol, it is possible to anti-blood protein adsorbs, thus extending blood circulation time, has good water solublity and stability.This nano-particle is all with biodegradable polyamino acid, and oligomeric ethylene glycol is construction unit, good biocompatibility, in vivo degradable, and catabolite can pass through kidney and directly get rid of external, harmless.
Accompanying drawing explanation
Fig. 1 is the proton nmr spectra spectrogram of the reversible nanogel of surface charge that embodiment 23 prepares；
Fig. 2 is the transmission electron microscope picture of the reversible nanogel of surface charge that embodiment 23 prepares；
Fig. 3 is that the surface potential of the reversible nanogel of surface charge prepared in embodiment 23 is with pH value change curve；
Fig. 4 is the cumulative release curve of medicament-carried nano gel prepared in embodiment 31；
Fig. 5 is the cytotoxicity experiment result to B16F10 cell of the medicament-carried nano gel described in embodiment 31；
Fig. 6 is the tumor suppression curve of the medicament-carried nano gel described in embodiment 31.
Detailed description of the invention
The preparation method that the invention provides the reversible nanogel of a kind of surface charge, comprises the following steps:
A) initiator and the Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride with morpholine group is dissolved in organic solvent and reacts, obtain mixed solution；
B) there is the compound of formula (I) structure, the compound with formula (II) structure and step A) mixed solution that obtains mixes, reacts, obtain reactant liquor；
C) by step B) reactant liquor that obtains mixes with organic solvent, filters, obtain the reversible nanogel of surface charge；
First initiator and Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride are dissolved in organic solvent and react by the present invention, obtain mixed solution.
Wherein, described initiator is with morpholine group, it is preferred to the one in compound shown in formula (101)～(104),
Nanogel surface provided by the invention, with morpholine group, is electric neutrality when the pH of blood, and is electropositive at tumor tissues, thus extending blood circulation time and making described nanoparticle easily by tumor tissues endocytosis.
In the present invention, described Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride has formula (III) structure,
Wherein n is the degree of polymerization, and the span of described n is preferably 1≤n≤5.
The source of described Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride is not particularly limited by the present invention, can be commercially available prod, it is also possible to prepare voluntarily according to the preparation method of Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride well known to those skilled in the art.
In the present invention, described Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride is preferably prepared in accordance with the following methods:
Pidolidone and oligomeric ethylene glycol monomethyl ether are carried out condensation reaction, obtains Pidolidone oligomeric ethylene glycol monomethyl ether ester；
Described Pidolidone oligomeric ethylene glycol monomethyl ether ester is carried out condensation reaction with double; two (trichloromethyl) carbonic esters, obtains Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride.
First, in anhydrous conditions, Pidolidone and oligomeric ethylene glycol monomethyl ether are carried out condensation reaction, obtains Pidolidone oligomeric ethylene glycol monomethyl ether ester.
Wherein, described oligomeric ethylene glycol monomethyl ether has formula (IV) structure:
Wherein n is the degree of polymerization, and the span of described n is preferably 1≤n≤5.
Pidolidone is mixed by the present invention with the stirring of oligomeric ethylene glycol monomethyl ether, obtains mixed liquor, drips concentrated sulphuric acid, react while stirring in described mixed liquor.Reaction regulates solution to neutral after terminating, be centrifuged and obtain solid.After described solids with methanol is dissolved, pour in isopropanol, be centrifuged and obtain solid, vacuum drying, obtain Pidolidone oligomeric ethylene glycol ester.The mol ratio of wherein said Pidolidone and oligomeric ethylene glycol monomethyl ether is preferably 1:1～10, more preferably 1:2～5, it is most preferred that for 1:5.Wherein the response time is preferably 12～36h, more preferably 12～24h, it is most preferred that for 24h.Wherein reaction temperature is preferably 20～30 DEG C, it is most preferred that be 25 DEG C.
In anhydrous conditions, the Pidolidone oligomeric ethylene glycol monomethyl ether ester obtained mixes in organic solvent with double; two (trichloromethyl) carbonic esters, carries out condensation reaction.
The mol ratio of described Pidolidone oligomeric ethylene glycol monomethyl ether ester and double; two (trichloromethyl) carbonic esters is preferably 1:(0.1～1.2), more preferably 1:(0.3～1), it is most preferred that for 1:(0.5～0.8).Described organic solvent is preferably oxolane.Temperature when described Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride mixes with double; two (trichloromethyl) carbonic esters is preferably 10 DEG C～40 DEG C, it is more preferably 15 DEG C～35 DEG C, it most preferably is 20 DEG C～30 DEG C, described reaction temperature is preferably 30 DEG C～80 DEG C, it is more preferably 35 DEG C～70 DEG C, it is most preferred that being 40 DEG C～60 DEG C, described condensation reaction time is preferably 0.1 hour～5 hours, it is more preferably 0.15 hour～3 hours, more preferably 0.2 hour～2 hours.
After condensation reaction terminates, the reactant liquor obtained preferably is settled with petroleum ether, the precipitum obtained is separated, obtain separating product.By described separation product washing, recrystallization, dry, obtain Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride.The washing of described separation product, recrystallization and dry method are not had particular restriction, method well known to those skilled in the art by the present invention.
Being dissolved in organic solvent by the Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride obtained and described initiator, the stirred under argon at nitrogen reacts.The mol ratio of described initiator and Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride is preferably 1:(5～50), more preferably 1:(5～25), it is most preferred that be 1:10.Described organic solvent is preferably N ' dinethylformamide, dioxane or chloroform, more preferably N ' dinethylformamide or dioxane, it is most preferred that for N ' dinethylformamide.The described quality of Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride and the volume ratio of organic solvent are preferably 1g:(1～20) mL, more preferably 1g:(3～15) mL, it is most preferred that for 1g:(5～10) mL.Described reaction temperature is preferably 15～50 DEG C, more preferably 20～40 DEG C, it is most preferred that be 25～35 DEG C.The described response time elects 48 hours～168 hours as, more preferably 72 hours～120 hours, it is most preferred that for 48d.
Reaction obtains mixed solution after terminating.The compound with formula (I) structure, the compound with formula (II) structure are mixed by the present invention with mixed solution obtained above, react.
Wherein, the compound with formula (I) structure is CYSTINE-N-ring inner-acid anhydride, containing disulfide bond in its molecular structure, described nanogel biocatalytic particle forms nanogel structure by disulfide bond crosslinking, in the enrichment of tumor locus targeting and by tumor cell endocytosis, can fast fracture under disulfide bond homoglutathion concentration in tumor cell, it is achieved the medicine intelligence release of inside tumor cells, reach to suppress the effect of tumor
The source of the described compound with formula (I) structure is not had particular restriction by the present invention, it is possible to for commercially available prod, it is also possible to the preparation method for those skilled in the art's resin is prepared voluntarily.In the present invention, the compound described in formula (I) structure is preferably prepared as follows:
CYSTINE is carried out condensation reaction with double; two (trichloromethyl) carbonic esters, obtains CYSTINE-N-ring inner-acid anhydride.
Concrete, in anhydrous conditions, with CYSTINE described in organic solvent dissolution, in organic solvent, CYSTINE carries out condensation reaction with double; two (trichloromethyl) carbonic esters, obtains CYSTINE-N-ring inner-acid anhydride.The mol ratio of described CYSTINE and double, two (trichloromethyl) carbonic esters is preferably 1:(0.1～1.2), it is more preferably 1:(0.3～1), it most preferably is 1:(0.5～0.8), described organic solvent is preferably oxolane, temperature when described CYSTINE-N-ring inner-acid anhydride mixes with double, two (trichloromethyl) carbonic esters is preferably 10 DEG C～40 DEG C, it is more preferably 15 DEG C～35 DEG C, it most preferably is 20 DEG C～30 DEG C, described reaction temperature is preferably 30 DEG C～80 DEG C, it is more preferably 35 DEG C～70 DEG C, it most preferably is 40 DEG C～60 DEG C, described condensation reaction time is preferably 0.1 hour～5 hours, it is more preferably 0.15 hour～3 hours, it is more preferably 0.2 hour～2 hours.
After condensation reaction terminates, the reactant liquor that obtains preferably being settled with petroleum ether, the precipitum that will obtain separates, then by the separation product washing obtained, recrystallization, dry, obtain CYSTINE-N-ring inner-acid anhydride.The washing of described separation product, recrystallization and dry method are not had particular restriction, method well known to those skilled in the art by the present invention.
In the present invention, the compound with formula (II) structure is L-phenylalanine-N-ring inner-acid anhydride.
The source of the described compound with formula (II) structure is not had particular restriction by the present invention, it is possible to for commercially available prod, it is also possible to the preparation method for those skilled in the art's resin is prepared voluntarily.In the present invention, the compound described in formula (II) structure is preferably prepared as follows:
L-phenylalanine is carried out condensation reaction with double; two (trichloromethyl) carbonic esters, obtains L-phenylalanine-N-ring inner-acid anhydride.
Concrete, in anhydrous conditions, with L-phenylalanine described in organic solvent dissolution, in organic solvent, L-phenylalanine carries out condensation reaction with double; two (trichloromethyl) carbonic esters, obtains the compound with formula (II) structure, i.e. L-phenylalanine-N-ring inner-acid anhydride.The mol ratio of described L-phenylalanine and double, two (trichloromethyl) carbonic esters is preferably 1:(0.1～1.2), it is more preferably 1:(0.3～1), it most preferably is 1:(0.5～0.8), described organic solvent is preferably oxolane, temperature when described L-phenylalanine mixes with double, two (trichloromethyl) carbonic esters is preferably 10 DEG C～40 DEG C, it is more preferably 15 DEG C～35 DEG C, it most preferably is 20 DEG C～30 DEG C, described reaction temperature is preferably 30 DEG C～80 DEG C, it is more preferably 35 DEG C～70 DEG C, it most preferably is 40 DEG C～60 DEG C, described condensation reaction time is preferably 0.1 hour～5 hours, it is more preferably 0.15 hour～3 hours, it is more preferably 0.2 hour～2 hours.
After condensation reaction terminates, the reactant liquor obtained preferably settles with petroleum ether, the precipitum that obtain is separated, then by the separation product washing obtained, recrystallization, dry, obtain L-phenylalanine-N-ring inner-acid anhydride.The washing of described separation product, recrystallization and dry method are not had particular restriction, method well known to those skilled in the art by the present invention.
The compound with formula (I) structure prepared above-mentioned, the compound with formula (II) structure mix with the mixed solution obtained, and react, obtain reactant liquor.
Concrete, compound, the compound with formula (II) structure and the mixed solution obtained will with formula (I) structure are mixed, stirring reaction in a nitrogen atmosphere.
Wherein, described initiator is preferably 1:(2～20 with the mol ratio of the compound with formula (I) structure), more preferably 1:(5～10), it is most preferred that be 1:10.The mol ratio of described initiator and the compound with formula (II) structure is preferably 1:(2～20), more preferably 1:(5～10), it is most preferred that be 1:10.Described reaction temperature is preferably 15～50 DEG C, more preferably 20～40 DEG C, it is most preferred that be 25～35 DEG C.The described response time is preferably 48 hours～168 hours, more preferably 72 hours～120 hours, it is most preferred that for 48d.
Reaction obtains reactant liquor after terminating.Described reactant liquor is mixed with organic solvent, filters, obtain the reversible nanogel of surface charge.Described organic solvent is preferably ethanol, after described reactant liquor mixes with ethanol, forms precipitate, after being filtered by described precipitate, carries out vacuum drying, obtain the reversible nanogel of surface charge.Described filtration and vacuum drying method are not had particular restriction by the present invention, well known to a person skilled in the art filtration and vacuum drying method.
Present invention also offers the reversible nanogel of surface charge that a kind of above-mentioned preparation method prepares.The reversible nanogel surface of surface charge provided by the invention with phenylboric acid group and has reduction response, it is possible to prepare the reversible nanogel medicine carrying granule of surface charge with antitumor drug.
Present invention also offers a kind of reversible nanogel medicine carrying granule of surface charge, the reversible nanogel of surface charge that this nanogel medicine carrying granule is prepared by antitumor drug and above-mentioned preparation method forms.The preparation method of the reversible nanogel medicine carrying granule of described surface charge is not had particular restriction by the present invention, and in the present invention, the reversible nanogel medicine carrying granule of described surface charge is preferably prepared as follows
The above-mentioned reversible nanogel of the surface charge prepared and antitumor drug are dissolved in organic solvent, mix homogeneously.Add isopyknic deionized water, stirring, dialysis, lyophilization, obtain the reversible nanogel medicine carrying granule of surface charge.Described organic solvent is preferably N ' dinethylformamide, dimethyl sulfoxide, more preferably dimethyl sulfoxide.The mode of described stirring and dialysis is not had particular restriction by the present invention, well known to a person skilled in the art stirring and the mode of dialysis.The time of described stirring is preferably 8～24h, is more preferably 8～16h, it is most preferred that be 8h.Wherein, the temperature of described dialysis is preferably 4～20 DEG C, more preferably 4～8 DEG C, it is most preferred that be 4 DEG C.The time of described dialysis is preferably 4～12h, more preferably 4～8h, it is most preferred that for 8h.The mass ratio of the reversible nanogel of described surface charge and antitumor drug is preferably 10:(10～1), more preferably 10:(8～3), it is most preferred that for 10:(6～4).Described antitumor drug is selected from amycin, epirubicin, Perarubicin, paclitaxel, Docetaxel, cisplatin, carboplatin, oxaliplatin, bortezomib, camptothecine or shikonin.
Nanogel medicine carrying particle surface provided by the invention, with morpholine group, is electric neutrality when the pH of blood, and is electropositive at tumor tissues, thus extending blood circulation time and making described nanoparticle easily by tumor tissues endocytosis.And, this nanogel biocatalytic particle forms nanogel structure by disulfide bond crosslinking, nanogel medicine carrying granule is obtained after medicine carrying, described medicine carrying granule is in the enrichment of tumor locus targeting and by tumor cell endocytosis, can fast fracture under disulfide bond homoglutathion concentration in tumor cell, realize the medicine intelligence release of inside tumor cells, reach to suppress the effect of tumor.Additionally, this nanogel medicine carrying granule has the shell of oligomeric ethylene glycol, it is possible to anti-blood protein adsorbs, thus extending blood circulation time, has good water solublity and stability.This nano-particle is all with biodegradable polyamino acid, and oligomeric ethylene glycol is construction unit, good biocompatibility, in vivo degradable, and catabolite can pass through kidney and directly get rid of external, harmless.
In order to be further appreciated by the present invention; below in conjunction with embodiment, a kind of reversible nanogel of surface charge provided by the invention and preparation method thereof and a kind of reversible nanogel medicine carrying granule of surface charge being illustrated, protection scope of the present invention is not limited by the following examples.
The preparation of embodiment 1～5L-glutamic acid oligomeric ethylene glycol ester
The glycol monoethyl ether of 20mL, the diethylene glycol monomethyl ether of 40mL, the triethylene glycol monomethyl ether of 60mL, the TEG monomethyl ether of 80mL and the five ethylene glycol monomethyl ether of 100mL are mixed with the stirring of 10gL-glutamic acid respectively, drips concentrated sulphuric acid, react while stirring.After reaction terminates, adjustment solution, to neutral, be centrifuged and obtain solid, after described solids with methanol dissolves, then pours in isopropanol, is centrifuged and obtains solid, vacuum drying, obtains Pidolidone oligomeric ethylene glycol monomethyl ether ester.Concrete yield is in Table 1, and table 1 is the yield of the Pidolidone oligomeric ethylene glycol monomethyl ether ester that embodiment 1～5 prepares.
The yield (yield please be supplement) of the Pidolidone oligomeric ethylene glycol monomethyl ether ester that table 1 embodiment 1～5 prepares
| Product | Yield/% | |
| Embodiment 1 | Pidolidone glycol monoethyl ether ester | 81.6 |
| Embodiment 2 | Pidolidone diethylene glycol monomethyl ether ester | 85.4 |
| Embodiment 3 | Pidolidone triethylene glycol monomethyl ether ester | 87.1 |
| Embodiment 4 | Pidolidone TEG monomethyl ether ester | 84.9 |
| Embodiment 5 | Pidolidone five ethylene glycol monomethyl ether ester | 89.3 |
The preparation of embodiment 6～10L-glutamic acid oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride
Pidolidone oligomeric ethylene glycol monomethyl ether ester described in 1g embodiment 1～5 is mixed under 25 DEG C of conditions with double; two (trichloromethyl) carbonic ester of 0.6g respectively, add oxolane, heat to 50 DEG C of reaction 2h, after reaction terminates, reactant mixture settled in excessive petroleum ether, separate, wash, recrystallization and obtain Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride after drying.Concrete yield is in Table 2, and table 2 is the yield of the Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride that embodiment 6～10 prepares.
The yield (asking the yield in supplementary table 2) of the Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride that table 2 embodiment 6～10 prepares
| Product | Yield/% | |
| Embodiment 6 | Pidolidone glycol monoethyl ether ester-N-ring inner-acid anhydride | 90.5 |
| Embodiment 7 | Pidolidone diethylene glycol monomethyl ether ester-N-ring inner-acid anhydride | 94.3 |
| Embodiment 8 | Pidolidone triethylene glycol monomethyl ether ester-N-ring inner-acid anhydride | 92.7 |
| Embodiment 9 | Pidolidone TEG monomethyl ether ester-N-ring inner-acid anhydride | 96.3 |
| Embodiment 10 | Pidolidone five ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride | 95.2 |
The preparation of poly-(the Pidolidone glycol monoethyl ether ester) of embodiment 11～15 different molecular weight
Weigh the N-aminomethyl morpholine shown in 1g formula (101); weigh 3.8g more respectively; 7.6g, 15.2g, 30.4g; 38gL-glutamic acid glycol monoethyl ether ester-N-ring inner-acid anhydride; joining in reaction bulb, nitrogen atmosphere is protected, and adds 100mLN ' dinethylformamide; reaction 72h, is gathered the solution of (Pidolidone glycol monoethyl ether ester).
The preparation of poly-(the Pidolidone oligomeric ethylene glycol monomethyl ether ester) of the different Pidolidone oligomeric ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride chain link of embodiment 16～19
Weigh the N-2-aminoethyl morpholine shown in 1g formula (102); weigh the Pidolidone diethylene glycol monomethyl ether ester-N-ring inner-acid anhydride of 9g more respectively; 10.4g Pidolidone triethylene glycol monomethyl ether ester-N-ring inner-acid anhydride; 14.2g Pidolidone TEG monomethyl ether ester-N-ring inner-acid anhydride; 15.8g Pidolidone five ethylene glycol monomethyl ether ester-N-ring inner-acid anhydride; join in reaction bulb; nitrogen atmosphere is protected; add 100mLN ' dinethylformamide; reaction 72h, obtains the solution of different poly-(Pidolidone oligomeric ethylene glycol monomethyl ether ester).
Poly-(the Pidolidone oligomeric ethylene glycol monomethyl ether ester) that the different initiator of embodiment 20～22 causes
Initiator shown in initiator shown in 1g formula (102), 1.1g formula (103) and the initiator shown in 1.1g formula (104) are mixed with 20.4gL-glutamic acid triethylene glycol monomethyl ether ester-N-ring inner-acid anhydride respectively; join in reaction bulb; nitrogen atmosphere is protected; add 100mLN ' dinethylformamide; reaction 72h, obtains the solution of different poly-(Pidolidone oligomeric ethylene glycol monomethyl ether ester).
The preparation of the polyamino acid nanogel of the different degree of cross linking of embodiment 23～26
By the L-phenylalanine-N-ring inner-acid anhydride of 0.57g respectively with 0.48g, CYSTINE-N-ring inner-acid anhydride the mix homogeneously of 0.95g, 1.43g, 1.90g, join in the solution of poly-(the Pidolidone oligomeric ethylene glycol monomethyl ether ester) described in embodiment 17, stirring reaction 3 days under nitrogen atmosphere.Being poured into by solution after reaction in 100mL absolute ether, sucking filtration takes solid, vacuum drying, obtains the reversible nanogel of surface charge.The embodiments of described nanogel is in Table 3, and table 3 is the sign of the reversible nanogel of surface charge of embodiment 23～26 preparation.
The surface charge of table 3 embodiment 23～26 preparation can change the sign of nanogel
| Embodiment | N(Glu-OEG3) | N(Phe) | N(Cys) | Molecular weight | Productivity (%) |
| 23 | 10 | 2.5 | 2.5 | 3630 | 97.2 |
| 24 | 10 | 2.5 | 5 | 4150 | 96.4 |
| 25 | 10 | 2.5 | 7.5 | 4680 | 97.1 |
| 26 | 10 | 2.5 | 10 | 5210 | 98.9 |
In table 3, N (Glu-OEG3), N (Phe), N (Cys) represents the ratio of poly-(Pidolidone triethylene glycol monomethyl ether ester) chain number and initiator molal quantity respectively；Molecular weight is the ratio of nanogel molecular weight and initiator number.
The reversible nanogel of surface charge embodiment 23 prepared carries out elementary analysis, and result is in Table 4, and table 4 is the elementary analysis table of the reversible nanogel of surface charge that embodiment 23 prepares.
The elementary analysis table of the reversible nanogel of surface charge that table 4 embodiment 23 prepares
| N (%) | C (%) | H (%) | S (%) |
| 7.50 | 52.77 | 6.645 | 4.917 |
As shown in Table 4, comprising required element in this nanogel, product successfully synthesizes.
The reversible nanogel of surface charge embodiment 23 prepared carries out nuclear magnetic resonance spectroscopy, and result is shown in the proton nmr spectra spectrogram that Fig. 1, Fig. 1 are the reversible nanogel of surface charge that embodiment 23 prepares.
The reversible nanogel of surface charge embodiment 23 prepared carries out transmission electron microscope test, and result is shown in the transmission electron microscope picture that Fig. 2, Fig. 2 are the reversible nanogel of surface charge that embodiment 23 prepares.
The reversible nanogel of surface charge embodiment 23 prepared is at pH7.4, pH7.2, pH7.0, pH6.8, surface potential test experiments is carried out when pH6.4 and pH6.0, concrete outcome see Fig. 3, Fig. 3 be in embodiment 23 surface potential of the reversible nanogel of surface charge of preparation with pH value change curve.From the figure 3, it may be seen that this nanogel carrier can occur electric charge to change when tumor organization pH, thus promoting cell endocytic.
The preparation of the polyamino acid nanogel of embodiment 27～30 not Homophenylalanine chain link
Respectively by 0.57g, L-phenylalanine-N-ring the inner-acid anhydride of 1.14g, 1.71g, 2.28g is mixed homogeneously with the CYSTINE-N-ring inner-acid anhydride of 1.90g, join in the solution of poly-(the Pidolidone oligomeric ethylene glycol monomethyl ether ester) described in embodiment 17, stirring reaction 72h under nitrogen atmosphere.Being poured into by solution after reaction in 100mL absolute ether, sucking filtration takes solid, vacuum drying, obtains the reversible nanogel of surface charge.Embodiments is in Table 5, and table 5 is the sign that surface charge prepared by embodiment 27～30 can change nanogel.
The surface charge of table 5 embodiment 27～30 preparation can change the sign of nanogel
| Embodiment | N(Glu-OEG3) | N(Phe) | N(Cys) | Molecular weight | Productivity (%) |
| 27 | 10 | 2.5 | 10 | 5210 | 97.5 |
| 28 | 10 | 5 | 10 | 5570 | 94.2 |
| 29 | 10 | 7.5 | 10 | 5940 | 98.7 |
| 30 | 10 | 10 | 10 | 6300 | 95.1 |
In table 5, N (Glu-OEG3), N (Phe), N (Cys) represents the ratio of poly-(Pidolidone triethylene glycol monomethyl ether ester) chain number and initiator molal quantity respectively；Molecular weight is the ratio of nanogel molecular weight and initiator number.
The preparation of the medicament-carried nano gel of the different drug loading of embodiment 31～35
Weigh the nanogel of preparation in 5 parts of 100mg embodiments 23 and be dissolved in 10mL dimethyl sulfoxide with 10mg, 20mg, 40mg, 60mg, 80mg amycin respectively, stir 12h.Add 10mL deionized water, stir 24h, dialysis, lyophilizing, obtain the reversible nanogel medicine carrying granule of surface charge.
Medicament-carried nano gel embodiment 31 prepared carries out amycin accumulation release test, concrete, by medicament-carried nano gel respectively when the phosphate buffered solution of the phosphate buffered solution of pH5.5, the phosphate buffered solution of pH6.8, the phosphate buffered solution of pH7.4 and 10mM glutathion, test the accumulation burst size of 1h, 2h, 4h, 6h, 10h, 12h, 24h, 36h, 48h and 72h amycin, concrete outcome is shown in the cumulative release curve that Fig. 4, Fig. 4 are medicament-carried nano gel prepared in embodiment 31.In Fig. 4, ■ is medicament-carried nano gel accumulation release profiles in the phosphate buffered solution of pH7.4, ● for medicament-carried nano gel accumulation release profiles in the phosphate buffered solution of pH6.8, ▲ for medicament-carried nano gel accumulation release profiles in the phosphate buffered solution of pH5.5For medicament-carried nano gel accumulation release profiles in 10mM glutathione solution.
The preparation of the nanogel kernel of embodiment 36～45 load different pharmaceutical
Weigh in 10 parts of 100mg embodiments 23 the nanogel kernel of preparation respectively with 20mg epirubicin, Perarubicin, paclitaxel, Docetaxel, cisplatin, carboplatin, oxaliplatin, bortezomib, camptothecine, shikonin is dissolved in 10mL dimethyl sulfoxide, stirs 12h.It is separately added into 10mL deionized water, stirs 24h, dialysis, lyophilizing, obtain medicine carrying kernel.
Embodiment 46 inhibition rate of tumor cell characterizes:
B16F10 cell is uniformly planted in 96 orifice plates, it is divided into 6 groups, often organize 7 holes, every porocyte number is about 7000, respectively with the DMEM culture medium of 3 groups of pH7.4, the DMEM culture medium of one group of pH=6.5, one group of DMEM culture medium containing glutathion, one group of DMEM culture medium culturing containing butyryl Asia cyclic imides, culture volume is 200 μ L.
Then it is 10,5,2.5,1.25,0.625,0.3125 and 0.1562mgmL by one group of concentration-1Amycin join in the DMEM culture medium orifice plate of one of which pH=7.4.Medicine carrying adriamycin nano gel particle embodiment 31 prepared again is divided into into 4 groups, and often group concentration is diluted successively is 10,5,2.5,1.25,0.625,0.3125 and 0.1562mgmL-1It is added separately to the DMEM culture medium of pH=6.5, the DMEM culture medium of pH=7.4, DMEM culture medium containing glutathion and containing in the orifice plate of the DMEM culture medium of butyryl Asia cyclic imides, the DMEM culture medium of one group of pH7.4 is not loaded with adriamycin nano gel and free amycin as a control group, again cultivates 24 hours.
Cultivation sucks culture medium after terminating, and by the solution-treated containing tetrazolium bromide, tests its absorption value in 490 nanometers.Cell survival rate uses below equation to calculate:
Concrete outcome is shown in the cytotoxicity experiment result to B16F10 cell that Fig. 5, Fig. 5 are the medicament-carried nano gel described in embodiment 31.In Fig. 5, ■ is medicament-carried nano gel cytotoxicity experiment result to B16F10 cell containing glutathion when, ● for medicament-carried nano gel cytotoxicity experiment result to B16F10 cell when pH6.5, ▲ for medicament-carried nano gel cytotoxicity experiment result to B16F10 cell containing butyryl Asia cyclic imides whenFor medicament-carried nano gel cytotoxicity experiment result to B16F10 cell when pH7.4, ◆ for free amycin cytotoxicity experiment result to B16F10 cell when pH7.4.
Embodiment 47 tumor inhibition
Choose load B16F10 transplanted tumor, body weight is the male C57 Mus 18 of about 20g, is divided into three groups, often group 6, and respectively through tail vein injection saline, amycin, drug-loading nanoparticles, the doxorubicin dosages of its injection is 5mgkg-1.The group of wherein said injecting normal saline is matched group.
Nail measure of time its tumor major diameter and minor axis, calculate gross tumor volume and use below equation to calculate:
Gross tumor volume=major diameter × minor axis × minor axis/2.
After measuring 15 days, put to death.Result is shown in the tumor suppression curve that Fig. 6, Fig. 6 are the medicament-carried nano gel described in embodiment 31.In Fig. 6, ● for the tumor suppression curve of medicament-carried nano gel, ◆ for the tumor suppression curve of free amycin,Tumor suppression curve for matched group.
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. the preparation method of the reversible nanogel of surface charge, it is characterised in that comprise the following steps:
A) initiator and the Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride with morpholine group is dissolved in organic solvent and reacts, obtain mixed solution；
B) there is the compound of formula (I) structure, the compound with formula (II) structure and step A) mixed solution that obtains mixes, reacts, obtain reactant liquor；
C) by step B) reactant liquor that obtains mixes with organic solvent, filters, obtain the reversible nanogel of surface charge；
2. preparation method according to claim 1, it is characterised in that the described initiator with morpholine group one in formula (101)～(104)
3. preparation method according to claim 1, it is characterised in that described Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride has formula (III) structure,
Wherein n is the degree of polymerization, 1 < n≤5.
4. preparation method according to claim 1, it is characterised in that step A) described in organic solvent selected from N ' dinethylformamide, dioxane or chloroform, step C) described in organic solvent be ether.
5. preparation method according to claim 1, it is characterised in that the mol ratio of the described initiator with morpholine group and Pidolidone oligomeric ethylene glycol monomethyl ether-N-ring inner-acid anhydride is 1:(5～50)；Described is 1:(2～20 with the initiator of morpholine group with the mol ratio of the compound with formula (I) structure)；Described is 1:(2～20 with the initiator of morpholine group with the mol ratio of the compound with formula (II) structure).
6. preparation method according to claim 1, it is characterised in that step A) temperature of described reaction is 15～50 DEG C, the time of described reaction is 48 hours～168 hours.
7. preparation method according to claim 1, it is characterised in that step B) temperature of described reaction is 15～50 DEG C, the time of described reaction is 48 hours～168 hours.
8. the reversible nanogel of surface charge that the preparation method as described in claim 1～7 any one claim prepares.
9. the reversible nanogel medicine carrying granule of surface charge, it is characterised in that the reversible nanogel of surface charge prepared by antitumor drug and the preparation method as described in claim 1～7 any one claim forms.
10. the reversible nanogel medicine carrying granule of surface charge according to claim 9, it is characterized in that, described antitumor drug is selected from amycin, epirubicin, Perarubicin, paclitaxel, Docetaxel, cisplatin, carboplatin, oxaliplatin, bortezomib, camptothecine or shikonin.
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