CN107296790A - A kind of mixing carrier micelle based on polyphosphate and preparation method thereof and a kind of mixing carrier micelle of active targeting base group modification - Google Patents
A kind of mixing carrier micelle based on polyphosphate and preparation method thereof and a kind of mixing carrier micelle of active targeting base group modification Download PDFInfo
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- CN107296790A CN107296790A CN201710526772.2A CN201710526772A CN107296790A CN 107296790 A CN107296790 A CN 107296790A CN 201710526772 A CN201710526772 A CN 201710526772A CN 107296790 A CN107296790 A CN 107296790A
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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Abstract
The invention provides a kind of mixing carrier micelle based on polyphosphate and preparation method thereof and a kind of mixing carrier micelle of active targeting base group modification.The present invention constitutes composite micelle by polyphosphate PLA di-block copolymer and PEG-PLA di-block copolymer, passes through composite micelle of the hydrophobic interaction power formation with identical PLA hydrophobic inner core, different hydrophilic shell between PLA block.The mixing carrier micelle good hydrophilic property that the present invention is provided, with good stability, and easily carry out targeting modification, have internal long circulating effect concurrently and promote target cell endocytosis and promote the effect of intracellular insoluble drug release, mixing carrier micelle after targeting modification can reach the double effect of passive target and active targeting, so as to further improve the therapeutic effect of tumour.
Description
Technical field
The present invention relates to the technical field of bio-medical material, more particularly to a kind of mixing based on polyphosphate carries medicine glue
Beam and preparation method thereof and a kind of mixing carrier micelle of active targeting base group modification.
Background technology
Polymer micelle is a kind of new nanometer medicine-carried system that grew up in recent years, is usually by amphipathic two block
(hydrophilic-hydrophobic) polymer or three block (hydrophilic-hydrophobic-hydrophilic) polymer self assembles are constituted, in addition also have some by
The hydrophilic-hydrophobic polymer of graft type or the Hydrophilic ionic type copolymer of ionic are formed.
Active targeting is primarily referred to as assigning the ability that medicine or its carrier are actively combined with target, and Main Means include will be anti-
The probe molecule that body, polypeptide, sugar chain, aptamer etc. can be specifically bound with target molecules passes through chemically or physically method
Medicine or its carrier surface are coupled to, so as to realize cancer target effect.
At present, hydrophilic chain is the most frequently used carrier micelle for the micella that PEG (polyethylene glycol) amphipathic nature polyalcohol is constituted,
PEG has the advantages such as good long circulating action, low immunogenicity, good biocompatibility, but PEG is greatly suppressed simultaneously
Tumour cell reduces therapeutic effect to its endocytosis.Active targeting modification can increase tumour cell endocytosis amount, but PEG lacks
Few avtive spot, it is difficult to carry out targeting modification.Meanwhile, PEG is difficult to degrade in human body, into tumour cell after insoluble drug release delay
Slowly.
The content of the invention
In view of this, present invention aims at provide a kind of mixing carrier micelle based on polyphosphate and preparation method thereof
With a kind of mixing carrier micelle of active targeting base group modification.The parent of the mixing carrier micelle based on polyphosphate that the present invention is provided
It is aqueous good, and targeting modification is easily carried out, have internal long circulating effect concurrently and promote to annex medicine in promotion tumour cell in target cell
The effect of thing release, the mixing carrier micelle after active targeting modification has excellent " passive " target and " active " targeted double
Effect.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of mixing carrier micelle based on polyphosphate, including carrier and coated by the carrier
Hydrophobicity chemotherapeutics；The carrier includes polyphosphate-PLA di-block copolymer and the block of polyethylene glycol-polylactic acid two
Copolymer；
The polyphosphate-PLA di-block copolymer has structure shown in Formulas I：
In Formulas I, m=5~30, n=10~150.
It is preferred that, the PEG2000-PLA12500 and polyphosphate-PLA di-block copolymer
Mass ratio is (1~20):(1~5).
It is preferred that, the weight average molecular weight of PLA block is 2000 in the polyphosphate-PLA di-block copolymer
~20000；The weight average molecular weight of polyphosphate block is 2000~10000；
The weight average molecular weight of PLA block is 2000~20000 in the PEG2000-PLA12500；
The weight average molecular weight of polyethylene glycol block is 2000~10000.
It is preferred that, the mass ratio of the hydrophobicity chemotherapeutics and carrier is (0.1~2):10.
The invention provides the preparation method that carrier micelle is mixed described in such scheme, comprise the following steps：
By polyphosphate-PLA di-block copolymer, PEG2000-PLA12500 hydrophobicity chemotherapeutic
Thing, organic basic compound and organic solvent mixing, obtain oil phase；
Oil phase is added to the water and emulsified, emulsion is obtained；
Organic solvent in emulsion is removed, the mixing carrier micelle based on polyphosphate is obtained.
It is preferred that, the organic basic compound includes the one or more in triethylamine, ethylenediamine and pyridine.
It is preferred that, the mol ratio of the organic basic compound and hydrophobicity chemotherapeutics is (1~10):1.
It is preferred that, the organic solvent is the mixed solvent of chloralkane and alcohols.
The invention provides a kind of active targeting base group modification mixing carrier micelle, including the mixing load described in such scheme
Mixing carrier micelle and modify on mixing carrier micelle carrier prepared by preparation method described in medicine micella or such scheme
Active targeting group；
The active targeting group is coupled on the polyphosphate block of polyphosphate-PLA di-block copolymer；
The active targeting group is arginine-glycine-aspartic acid-phenylalanine peptide.
It is preferred that, the quality of the active targeting group is total to for the block of polyphosphate-PLA two in mixing carrier micelle
The 1%~20% of polymers and PEG2000-PLA12500 gross mass.
The invention provides a kind of mixing carrier micelle based on polyphosphate, including carrier and coated by the carrier
Hydrophobicity chemotherapeutics；The carrier includes polyphosphate-PLA di-block copolymer and the block of polyethylene glycol-polylactic acid two
Copolymer.The present invention constitutes multiple by polyphosphate-PLA di-block copolymer and PEG2000-PLA12500
Rubber alloy beam, is formed outside with identical PLA hydrophobic inner core, different hydrophilic by the hydrophobic interaction between PLA block
The composite micelle of shell, can overcome the shortcoming of single micella, and can retain the respective advantage of single micella.It is mixed that the present invention is provided
Carrier micelle good hydrophilic property is closed, with good stability, and targeting modification is easily carried out, has internal long circulating effect and rush concurrently
Target cell endocytosis and the effect for promoting intracellular insoluble drug release.Embodiment result shows, quiet using adriamycin as hydrophobicity chemotherapeutics
The arteries and veins injection present invention is mixed after carrier micelle 1h, and the doxorubicin concentration in mice serum is small apparently higher than the free adriamycin of injection
Mouse, illustrating the mixing carrier micelle of the present invention has excellent internal long circulating effect；To this hair by taking Bel-7402 cells as an example
The mixing carrier micelle of bright offer promotees target cell endocytosis and intracellular insoluble drug release is tested, as a result show in pH6.5 and
In the case of 7.4, Bel-7402 cells are to mixing the endocytosis amount of carrier micelle apparently higher than PEG-PLLA micellas.
The invention provides the preparation method that carrier micelle is mixed described in such scheme, the preparation method behaviour that the present invention is provided
Make simple, cost is low, easily carries out industrialized production.
Present invention also offers a kind of active targeting base group modification mixing carrier micelle, including the mixing described in such scheme
Mixing carrier micelle and modify on mixing carrier micelle carrier prepared by preparation method described in carrier micelle or such scheme
Active targeting group；The polyphosphate that the active targeting group is coupled at polyphosphate-PLA di-block copolymer is embedding
Duan Shang；The active targeting group is arginine-glycine-aspartic acid-phenylalanine peptide (RGDF).The present invention repaiies RDGF
Decorations form the mixing of active targeting base group modification and carry medicine on the polyphosphate block of polyphosphate-PLA di-block copolymer
Micella.Embodiment result shows that the RGDF in the mixing carrier micelle for the active targeting base group modification that the present invention is provided is in pH
It can be hidden, it is to avoid the phagocytosis of normal structure, be reached after tumor locus, tumour in PEG hydrated sheath under conditions of 7.4
The slightly sour environment at position enables RGDF to expose, and reaches the effect of active targeting, increases the endocytosis of target cell, and it is passive to be finally reached
Targeting and the double effect of active targeting, so as to further improve the therapeutic effect of tumour.
Brief description of the drawings
Fig. 1 is PAEEP prepared by the embodiment of the present invention 112-PLLA70The RGDF-PAEEP-PLLA's prepared with embodiment 81H-NMR nuclear magnetic spectrums；
Fig. 2 is PAEEP prepared by the embodiment of the present invention 112-PLLA70The RGDF-PAEEP-PLLA's prepared with embodiment 8
Infared spectrum；
Fig. 3 is the medicine of the embodiment of the present invention 2, mixing carrier micelle prepared by embodiment 9 and PEG-PLLA carrier micelles
In-vitro release curves figure；
Fig. 4 carries medicine for the mixing that the Bel7402 cells of flow cytomery are prepared to the embodiment of the present invention 2, embodiment 9
The endocytosis spirogram of micella and PEG-PLLA carrier micelles；
Fig. 5 carries for the mixing that the Bel7402 cells of flow cytomery are prepared to the embodiment of the present invention 2, embodiment 10
The endocytosis spirogram of medicine micella and PEG-PLLA carrier micelles；
Fig. 6 carries for the mixing that the Bel7402 cells of flow cytomery are prepared to the embodiment of the present invention 2, embodiment 11
The endocytosis spirogram of medicine micella and PEG-PLLA carrier micelles；
Fig. 7 is that the mixing that the macrophages of mouse RAW 264.7 are prepared to embodiment 2, embodiment 9, embodiment 11 carries medicine glue
The endocytosis spirogram of beam；
Medicine of the mixing carrier micelle that Fig. 8 is embodiment 2, prepared by embodiment 9, embodiment 11 in the intracellular 12h of Bel7402
Thing release conditions figure；
Drug-time curve of the mixing carrier micelle that Fig. 9 is embodiment 2, prepared by embodiment 9, embodiment 11 in Mice Body；
Figure 10 is the mixing carrier micelle for embodiment 2, embodiment 9, embodiment 11 preparation for containing DIR in tumor-bearing mice body
Interior distribution situation figure；
The mixing carrier micelle that Figure 11 is embodiment 2, prepared by embodiment 9, embodiment 11 is in tumor-bearing mice body after 24h
Tissue distribution patterns figure；
Figure 12 is that the mixing carrier micelle that tumor-bearing mice is prepared using embodiment 2, embodiment 9, embodiment 11 is treated 21 days
Interior tumor volume change curve map；
Figure 13 is that the mixing carrier micelle that tumor-bearing mice is prepared using embodiment 2, embodiment 9, embodiment 11 is treated 21 days
Interior changes of weight curve map；
Figure 14 is that the mixing carrier micelle that tumor-bearing mice is prepared using embodiment 2, embodiment 9, embodiment 11 is treated 21 days
The weight chart of tumour afterwards；
Figure 15 is that the mixing carrier micelle that tumor-bearing mice is prepared using embodiment 2, embodiment 9, embodiment 11 is treated 21 days
Tumour picture afterwards.
Embodiment
The invention provides a kind of mixing carrier micelle based on polyphosphate, including carrier and coated by the carrier
Hydrophobicity chemotherapeutics；The carrier includes polyphosphate-PLA di-block copolymer and the block of polyethylene glycol-polylactic acid two
Copolymer；
The polyphosphate-PLA di-block copolymer has structure shown in Formulas I：
In Formulas I, m=5~30, n=10~150.
The mixing carrier micelle that the present invention is provided includes carrier, and it is embedding that the carrier includes polyphosphate-PLA two
Section copolymer (PAEEP-PLLA), with structure shown in Formulas I.In the present invention, the span of the m is 5~30, is preferably
15~20；The span of the n is 10~150, preferably 60~140, more preferably 70；Gather breast in the PAEEP-PLLA
The weight average molecular weight of sour block is preferably 2000~20000, and more preferably 6000~8000；Polyphosphoric acid in the PAEEP-PLLA
The weight average molecular weight of ester block is preferably 2000~10000, more preferably 3000~7000, most preferably 3000~5000.
Source no particular/special requirement of the invention to polyphosphate-PLA di-block copolymer, using art technology
Known to personnel prepared by method, in a particular embodiment of the present invention, and the block of polyphosphate-PLA two is total to
The method of polymers prepares and preferably includes following steps：
(1) by PLA, 2- (N- (tertbutyloxycarbonyl) monoethanolamine) -2- oxygen -1,3,2- dioxaphospholane and organic
Solvent is mixed, and is carried out ring-opening polymerization under organo-metallic catalyst effect, is obtained intermediate product；
(2) intermediate product for obtaining the step (1) is mixed with organic solvent and acid reagent, and progress is deprotected anti-
Should, obtain the polyphosphate with structure shown in Formulas I-PLA di-block copolymer.
The present invention is by PLA (PLLA), 2- (N- (tertbutyloxycarbonyl) monoethanolamine) -2- oxygen -1,3,2- dioxies phosphorus heterocycle penta
Alkane (N-Boc-EAOP) and organic solvent mixing, carry out ring-opening polymerization under organo-metallic catalyst effect, obtain centre
Product (N-Boc-PAEEP-PLLA).In the present invention, the PLLA, N-Boc-EAOP, organic solvent and metal organic catalysis
The mass ratio of agent is preferably (1~2):1:(50~200):(0.1~0.5), more preferably 1:1:(100~150):(0.2~
0.3)。
The present invention originates without special restriction for the PLLA's, using PLLA cities well known to those skilled in the art
Sell commodity or the PLLA products prepared using method well known to those skilled in the art.
The present invention originates without special restriction for the N-Boc-EAOP's, using well known to those skilled in the art
The N-Boc-EAOP products that method is prepared.In the present invention, the N-Boc-EAOP is preferably according to following steps system
It is standby to obtain：
The chloro- 2- oxygen -1,3,2- dioxaphospholane of 2- and N- (tertbutyloxycarbonyl) monoethanolamine are entered in organic solvent
Row substitution reaction, obtains N-Boc-EAOP.
In the present invention, the chloro- 2- oxygen -1,3 of the 2-, 2- dioxaphospholane, N- (tertbutyloxycarbonyl) monoethanolamines and
The mol ratio of organic solvent is preferably 1：(0.9~1.1)：(15~40), more preferably 1：1：(20~30).In the present invention,
The organic solvent is preferably aprotic organic solvent.The present invention does not have special for the species of the aprotic organic solvent
Limit, using aprotic organic solvent well known to those skilled in the art.In the present invention, the aprotic organic solvent
Preferably include tetrahydrofuran, ethyl acetate, dichloromethane or chloroform.
In the present invention, the temperature of the substitution reaction is preferably -4~0 DEG C；Time is preferably 12~24h, more preferably
14~20h, most preferably 16~18h.
Complete after the substitution reaction, the present invention is preferably post-processed the product of substitution reaction, obtains the N-
Boc-EAOP.In the present invention, the post processing preferably includes following steps：
The solvent in the product of substitution reaction is removed, obtained solid is dried, N-Boc-EAOP is obtained.
The present invention does not have special restriction for removing the mode of solvent, molten using removal well known to those skilled in the art
The technical scheme of agent.The product of the substitution reaction is preferably carried out rotary evaporation by the present invention, to remove solvent.The present invention
There is no special restriction for the mode of the drying, using drying mode well known to those skilled in the art.The present invention
It is preferred to use vacuum drying.In the present invention, the vacuum drying temperature is preferably 50~70 DEG C, more preferably 55~65
℃；Time is preferably 20~28h, more preferably 22~26h；Vacuum is preferably smaller than 0.05atm.
In the present invention, it is preferably aprotic organic solvent with the PLLA and the N-Boc-EAOP organic solvent mixed.
The present invention does not have special restriction for the species of the aprotic organic solvent, using non-matter well known to those skilled in the art
Sub- organic solvent.In the present invention, the aprotic organic solvent preferably includes tetrahydrofuran, ethyl acetate, dichloromethane
Alkane or chloroform.
The present invention does not have special restriction for the species of the organo-metallic catalyst, ripe using those skilled in the art
That knows is used for the organo-metallic catalyst that catalyzed ring opening polymerization reacts.In the present invention, the organo-metallic catalyst is excellent
Choosing includes organotin catalysts and/or organo aluminum catalyst.The present invention is not special for the species of the organotin catalysts
Restriction, using it is well known to those skilled in the art for catalyzed ring opening polymerization react organotin catalysts, such as octanoic acid
Stannous (Sn (Oct)2).The present invention does not have special restriction for the species of the organo aluminum catalyst, using art technology
The organo aluminum catalyst reacted known to personnel for catalyzed ring opening polymerization, such as aluminum isopropylate (Al (OiPr)3) or three
Aluminium isobutyl (iBu3Al)。
In the present invention, the temperature of the ring-opening polymerization is preferably 50~60 DEG C, more preferably 53~57 DEG C；Time
Preferably 3~12h, more preferably 5~10h, most preferably 6~8h.The ring-opening polymerization mild condition that the present invention is provided, institute
Need reaction temperature relatively low.
Complete after the ring-opening polymerization, the present invention is preferably post-processed the product of the ring-opening polymerization,
Obtain intermediate product.In the present invention, the post processing preferably includes following steps：
By the product precipitation, separation of solid and liquid, drying of the ring-opening polymerization, intermediate product is obtained.
The present invention does not have special restriction for the method for the precipitation, using it is well known to those skilled in the art can be by
The technical scheme that the product of the ring-opening polymerization is precipitated.Present invention preferably employs non-polar organic solvent to institute
The product for stating ring-opening polymerization is precipitated.The present invention does not have special limit for the species of the non-polar organic solvent
It is fixed, using non-polar organic solvent well known to those skilled in the art, such as ether or petroleum ether.In the present invention, it is described
The mass ratio of non-polar organic solvent and the product of the ring-opening polymerization is preferably (45~55)：1, more preferably (48~
52)：1.
Complete after the precipitation, the material obtained after being precipitated described in preferred pair of the present invention carries out separation of solid and liquid.The present invention is right
There is no special restriction in the mode of the separation of solid and liquid, using solid-liquid separation method well known to those skilled in the art.
The separation of solid and liquid is realized present invention preferably employs filtering.
Complete after the separation of solid and liquid, the solid obtained after separation of solid and liquid described in preferred pair of the present invention is dried, and obtains
Intermediate product.The present invention does not have special restriction for the mode of the drying, using drying well known to those skilled in the art
Mode.Present invention preferably employs vacuum drying.In the present invention, the vacuum drying temperature is preferably 50~70 DEG C,
More preferably 55~65 DEG C；Time is preferably 20~28h, more preferably 22~26h；Vacuum is preferably smaller than 0.05atm.
Obtain after intermediate product, the present invention mixes the intermediate product with organic solvent and acid reagent, carry out remove-insurance
Shield reaction, obtains the polyphosphate with structure shown in Formulas I-PLA di-block copolymer.In the present invention, the middle production
The mass ratio of thing, organic solvent and acid reagent is preferably 1：(10~20)：(0.5~1), more preferably 1：(13~17)：
(0.6~0.8).
In the present invention, the organic solvent mixed with the intermediate product and acid reagent is preferably non-proton organic molten
Agent.The present invention does not have special restriction for the species of the aprotic organic solvent, using well known to those skilled in the art
Aprotic organic solvent.In the present invention, the aprotic organic solvent preferably includes tetrahydrofuran, ethyl acetate, two
Chloromethanes or chloroform.
The present invention does not have special restriction for the species of the acid reagent, is used using well known to those skilled in the art
In progress deprotection reaction to slough the acid reagent of tertbutyloxycarbonyl Boc blocking groups, such as trifluoroacetic acid or hydrogen chloride.
In the present invention, the temperature of the deprotection reaction is preferably 20~40 DEG C, more preferably 25~35 DEG C；Specifically
, in embodiments of the invention, the deprotection reaction is carried out at room temperature, without heating without cooling.In the present invention,
The time of the deprotection reaction is preferably 3~6h, more preferably 4~5h.The deprotection reaction mild condition that the present invention is provided,
The deprotection reaction can be carried out at room temperature.
Complete after the deprotection reaction, the present invention is preferably post-processed the product of the deprotection reaction, is obtained
Polyphosphate-PLA the di-block copolymer with structure shown in Formulas I.In the present invention, the preferred bag of the post processing
Include following steps：
By the product precipitation, separation of solid and liquid, drying of the deprotection reaction, obtain described poly- with structure shown in Formulas I
Phosphate-PLA di-block copolymer.
The present invention does not have special restriction for the method for the precipitation, using it is well known to those skilled in the art can be by
The technical scheme that the product of the ring-opening polymerization is precipitated.Present invention preferably employs non-polar organic solvent to institute
The product for stating ring-opening polymerization is precipitated.The species of non-polar organic solvent of the present invention needed for for carrying out the precipitation
There is no special restriction, using non-polar organic solvent well known to those skilled in the art, such as ether or petroleum ether.At this
In invention, the mass ratio of the non-polar organic solvent and the product of the deprotection reaction is preferably (15~25)：1, it is more excellent
Elect as (18~22)：1.
Complete after the precipitation, the material obtained after being precipitated described in preferred pair of the present invention carries out separation of solid and liquid.The present invention is right
There is no special restriction in the mode of the separation of solid and liquid, using solid-liquid separation method well known to those skilled in the art.
The separation of solid and liquid is realized present invention preferably employs filtering.
Complete after the separation of solid and liquid, the solid obtained after separation of solid and liquid described in preferred pair of the present invention is dried, and obtains
Intermediate product.The present invention does not have special restriction for the mode of the drying, using drying well known to those skilled in the art
Mode.Present invention preferably employs vacuum drying.In the present invention, the vacuum drying temperature is preferably 40~60 DEG C,
More preferably 45~55 DEG C；Time is preferably 9~15h, more preferably 11~13h；Vacuum is preferably smaller than 0.05atm.
In the present invention, the carrier includes PEG2000-PLA12500 (PEG-PLLA).In the present invention
In, the weight average molecular weight of PLA block is preferably 2000~20000 in the PEG-PLLA, and more preferably 6000~8000；
The weight average molecular weight of polyethylene glycol block is preferably 2000~10000 in the PEG-PLLA, and more preferably 6000~8000.
In the present invention, the mass ratio of the PAEEP-PLLA and PEG-PLLA are preferably (1~20):(1~5), it is more excellent
Elect as (2~18):1, most preferably 16:1.
In the present invention, the structure of the PEG-PLLA is as shown in formula II：
In the present invention, the span of the p is preferably 10~150, and more preferably 50~100；The value model of the q
Enclose preferably 10~150, more preferably 50~100.
The present invention knows the PEG- in source using those skilled in the art to the PEG-PLLA no particular/special requirement in source
PLLA, it is specific such as commercial goods.
The present invention by control in two kinds of block copolymers the molecular weight of each block and the mass ratio of two kinds of copolymers come
Improve long circulating effect and target cell endocytosis effect of mixing carrier micelle.In the carrier for the mixing carrier micelle that the present invention is provided
Including polyphosphate-PLA di-block copolymer and PEG2000-PLA12500, by PLA block it
Between hydrophobic interaction power formation have identical PLA hydrophobic inner core, the composite micelle of different hydrophilic shell, mixing carry medicine
Micella can overcome that single polyethylene glycol-polylactic acid micella target cell endocytosis effect is poor, be difficult to the shortcoming of targeting modification
With single polyphosphate-PLA micella poorly water-soluble, the bad shortcoming of internal drug effect；And the hydrophilic outer shell of composite micelle
It is middle because having the acid-sensitive and quick characteristic of enzyme containing polyphosphate, it is easy to degrade so that medicine in the cell can quick release,
Reach the purpose that antineoplastic is efficiently conveyed.
In the present invention, the mass ratio of the hydrophobicity chemotherapeutics and carrier is preferably (0.1~2):10, more preferably
(0.5~1.8):10, most preferably 1.5:10.The present invention does not have particular/special requirement to the species of the hydrophobicity chemotherapeutics, makes
With hydrophobicity chemotherapeutics well known to those skilled in the art, specific such as doxorubicin hydrochloride, taxol, cis-platinum.
The invention provides the preparation method that carrier micelle is mixed described in such scheme, comprise the following steps：
By polyphosphate-PLA di-block copolymer, PEG2000-PLA12500, hydrophobicity chemotherapy
Medicine, organic basic compound and organic solvent mixing, obtain oil phase；
Oil phase is added to the water and emulsified, emulsion is obtained；
Organic solvent in emulsion is removed, the mixing carrier micelle based on polyphosphate is obtained.
The present invention is by polyphosphate-PLA di-block copolymer (PAEEP-PLLA), the block of polyethylene glycol-polylactic acid two
Copolymer (PEG-PLLA), hydrophobicity chemotherapeutics, organic basic compound and organic solvent mixing, obtain oil phase.In this hair
In bright, the mass ratio of the PEG-PLLA and PAEEP-PLLA are preferably (1~20):(1~5), more preferably (2~18):1,
Most preferably 16:1；The hydrophobicity chemotherapeutics and the mass ratio of PAEEP-PLLA, PEG-PLLA gross mass are preferably (0.1
~2):10, more preferably (0.5~1.8):10, most preferably 1.5:10；The organic basic compound preferably includes three second
One or more in amine, ethylenediamine and pyridine, more preferably triethylamine；The organic basic compound and hydrophobicity chemotherapeutic
The mol ratio of thing is (1~3):1, more preferably 2:1；The organic solvent is preferably the mixed solvent of chloralkane and alcohols；
The chloralkane is preferably chloroform or dichloromethane；The alcohols is preferably methanol or ethanol；The chloralkane and alcohol
Volume ratio is preferably (1~3):1, more preferably 2:1；In a particular embodiment of the present invention, the organic solvent is preferably chlorine
Imitative and methanol is with volume ratio 2:The solvent of 1 mixing；The mass ratio of organic solvent and PAEEP-PLLA, PEG-PLLA gross mass
Preferably (10~15):1, more preferably (12~13):1.
The present invention is to the PAEEP-PLLA, PEG-PLLA, hydrophobicity chemotherapeutics, organic basic compound and organic molten
The order by merging of agent does not have particular/special requirement, is mixed using random order, can make PAEEP-PLLA, PEG-PLLA, hydrophobicity
Medicine, organic basic compound is treated to be dissolved completely in organic solvent.
Obtain after oil phase, oil phase is added to the water by the present invention to be emulsified, and obtains emulsion.In the present invention, the water is excellent
The volume ratio for electing organic solvent in ultra-pure water, the water and oil phase as is preferably (5~15):1, more preferably 10:1.
Oil phase is preferably added drop-wise in water by the present invention to be emulsified, and the speed of the dropwise addition is preferably 0.05~0.1mL/s,
More preferably 0.06~0.08mL/s；The emulsification is preferably ultrasonic emulsification, and the power of the ultrasonic emulsification is preferably 200~
400W, more preferably 300W；The time of the ultrasonic emulsification is preferably 5~10min, more preferably 6~8min；The present invention's is super
Sound emulsification times since oil phase be added dropwise start when calculating；The present invention does not have particular/special requirement to the instrument that ultrasonic emulsification is used, and makes
It is specific such as cell crushing instrument with ultrasonic emulsification instrument well known to those skilled in the art.
Obtain after emulsion, the present invention removes the organic solvent in emulsion, obtain the mixing based on polyphosphate and carry medicine glue
Beam (PEG-PLLA/PAEEP-PLLA mixing carrier micelle).The present invention preferably makes the organic solvent in emulsion volatilize by stirring；
The rotating speed of the stirring is preferably 150~300rpm, more preferably 250~280rpm；The time of the stirring is preferably 24~
48h, more preferably 35~40h；The temperature of the stirring is preferably room temperature, without carrying out extra heating and cooling.
The carrier for the mixing carrier micelle that the present invention is provided includes PEG-PLLA and PAEEP-PLLA, wherein PAEEP-PLLA
PAEEP blocks in there is amino, easily carry out active targeting modification, active targeting modification after mixing carrier micelle tumour
Therapeutic effect can be further improved.
Present invention also offers a kind of active targeting base group modification mixing carrier micelle, including the mixing described in such scheme
The active targeting group of carrier micelle and modification on mixing carrier micelle carrier；The active targeting group is coupled at poly- phosphorus
On the polyphosphate block of acid esters-PLA di-block copolymer；The active targeting group is arginine-glycine-asparagus fern
Propylhomoserin-phenylalanine peptide (RGDF).In the present invention, the quality of the active targeting group is preferably and mixes in carrier micelle to gather
The 1%~20% of phosphate-PLA di-block copolymer and PEG2000-PLA12500 gross mass, it is more excellent
Elect 5%~15%, most preferably 6.5%~8.5% as, be further preferably 6.5%.
In the present invention, the active targeting group is coupled on PAEEP-PLLA polyphosphate block (PAEEP), this
Invention does not have particular/special requirement to the method that active targeting group is coupled on PAEEP blocks, is known using those skilled in the art
Coupling method.In a particular embodiment of the present invention, the method that active targeting group is coupled on PAEEP blocks
Preferably include following steps：
PAEEP-PLLA, RGDF, coupling agent and organic solvent are mixed, coupling reaction is carried out, RGDF modifications are obtained
PAEEP-PLLA(RGDF-PAEEP-PLLA)。
In the present invention, the mol ratio of amino is preferably (1~3) on PAEEP side chains in the RGDF and PAEEP-PLLA:
1, more preferably 2:1；The coupling agent is preferably 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC)
With mixture, the N of n-hydroxysuccinimide (NHS), N'- dicyclohexylcarbodiimides (DCC) and DMAP
(DMAP) mixture；EDC and NHS mol ratio is preferably 1 in the mixture of the EDC and NHS:1；The DCC and DMAP
Mixture in DCC and DMAP mol ratio be preferably 1:1；The mol ratio of the coupling agent and RGDF is preferably (5~12):1,
More preferably 10:1.
In the present invention, the organic solvent is preferably dimethyl sulfoxide (DMSO), or chloralkane and alcohol mixed solvent；It is described
Chloralkane is preferably chloroform or dichloromethane；The alcohol is preferably ethanol or methanol；The volume ratio of the chloralkane and alcohol
Preferably (2~3):1, more preferably 2.5:1；The mass ratio of organic solvent and PAEEP-PLLA, RGDF gross mass is preferred
For (5~10):1, more preferably (6~8):1.
In the present invention, the temperature of the coupling reaction is preferably room temperature, without carrying out extra heating and cooling；It is described
The time of coupling reaction is preferably 24~48h, more preferably 30~40h；The present invention preferably carries out being coupled instead under agitation
Should, the rotating speed of the stirring is preferably 150~300rpm, more preferably 200~250rpm.
After the completion of the coupling reaction, preferred pair reaction solution of the present invention is post-processed, and obtains RGDF-PAEEP-PLLA.
In the present invention, the post processing preferably includes following steps：
By reaction solution separation of solid and liquid, solid product is obtained；
The solid product is dialysed and is freeze-dried successively.
The present invention does not have particular/special requirement to the specific method of separation of solid and liquid, uses solid-liquid well known to those skilled in the art point
It is specific as filtered from method.
Obtain after solid product, the present invention is dialysed and is freeze-dried successively to the solid product.In the present invention,
The dialysis is preferably 800~1200 with the molecular cut off of bag filter, and more preferably 1000；The time of the dialysis is preferably
24~72h, more preferably 36~48h；The present invention removes small point of the RGDF not being coupled in product and coupling agent etc. by dialysing
Sub- impurity.
After the dialysis, dialysis product is freeze-dried by the present invention, obtains RGDF-PAEEP-PLLA.In the present invention
In, the time of the freeze-drying is preferably 24~48h, more preferably 30~40h；The freeze-drying is preferably vacuum refrigeration
Dry；The present invention does not have particular/special requirement to the vacuum of vacuum freeze drying, can keep carrying out freezing under vacuum conditions doing
It is dry.
In the present invention, the preparation method of the mixing carrier micelle of the active targeting base group modification and above-mentioned preparation mixing
The scheme of carrier micelle is consistent, differs only in and active targeting group is coupled on PAEEP-PLLA PAEEP blocks first,
Then RGDF-PAEEP-PLLA and PEG-PLLA is mixed again, emulsification is carried out and organic according to such scheme identical method
The removal of solvent.
The present invention on PAEEP-PLLA PAEEP blocks by being coupled RGDF, and the mixing for obtaining active targeting modification is carried
RGDF can be hidden in PEG hydrated sheaths by medicine micella, the mixing carrier micelle that the present invention is provided in neutral conditions, reduce monokaryon
The phagocytosis of macrophage system, and in the slightly sour environment of tumour, PAEEP side chain and RGDF protonations increase obtains RGDF
It is exposed, reaches the effect of active targeting, increase the endocytosis of target cell, is finally reached the dual of passive target and active targeting
Effect, so as to further improve the therapeutic effect of tumour.
With reference to embodiment to mixing carrier micelle based on polyphosphate for providing of the present invention and preparation method thereof and
A kind of mixing carrier micelle of active targeting base group modification is described in detail, but they can not be interpreted as to the present invention
The restriction of protection domain.
Embodiment 1
By chloro- 2- oxygen -1,3,2- dioxaphospholane 1mol, N- (tertbutyloxycarbonyl) the monoethanolamine 0.9mol of 2- and tetrahydrochysene
Furans 40mol is mixed, and substitution reaction 12h is carried out under the conditions of 0 DEG C, and rotary evaporation falls after solvent, is 0.03atm's in vacuum
Under the conditions of 60 DEG C vacuum drying 20h, obtain 2- (N- (tertbutyloxycarbonyl) monoethanolamine) -2- oxygen -1,3,2- dioxaphospholane
(N-Boc-EAOP)
(2) PLA, N-Boc-EAOP and tetrahydrofuran are mixed, under organo aluminum catalyst effect, in 60 DEG C of conditions
Lower progress ring-opening polymerization 3h, obtains intermediate product；
(3) intermediate product for obtaining the step (2) is mixed with tetrahydrofuran and trifluoroacetic acid, and progress is deprotected anti-
Should, the material obtained after then being terminated with ether to the deprotection reaction is precipitated, filtering, by obtained solid in vacuum
Spend and be dried in vacuo 15h for 40 DEG C under conditions of 0.03atm, obtain PAEEP12-PLLA70。
Synthetic route is as shown in formula a：
Embodiment 2
Doxorubicin hydrochloride 8.2mg, PEG are weighed respectively83-PLLA70(wherein the weight average molecular weight of PEG block is 5000,
The PAEEP that the weight average molecular weight of PLLA blocks is prepared for 5000) 60.2mg and embodiment 112-PLLA70(wherein PAEEP blocks
Weight average molecular weight is that the weight average molecular weight of 2000, PLLA blocks is 5000) 40.2mg, and 1mL chloroforms and 0.5mL methanol are dissolved in jointly
In the mixed solvent, and add 50 μ L triethylamine solutions by doxorubicin hydrochloride carry out desalination acid treatment, until completely dissolved, dropwise
Add in 15mL ultra-pure waters and carry out ultrasonic emulsification using cell crushing instrument simultaneously, keep power 200w, time 5min, obtain homogeneous
Stable emulsion；Emulsion is placed in room temperature 24h is stirred under 150rpm, volatilization removes organic solvent and obtains mixing carrier micelle.
Embodiment 3
PAEEP is prepared according to the method for embodiment 112-PLLA70；
Doxorubicin hydrochloride 9.6mg, PEG are weighed respectively83-PLLA70(wherein the weight average molecular weight of PEG block is 5000,
The weight average molecular weight of PLLA blocks is 5000) 71.4mg and PAEEP12-PLLA70(weight average molecular weight of wherein PAEEP blocks is
The weight average molecular weight of 2000, PLLA blocks is 5000) 30.5mg, and the mixed solvent of 1mL chloroforms and 0.5mL ethanol is dissolved in jointly
In, and 50 μ L triethylamine solutions are added by doxorubicin hydrochloride progress desalination acid treatment, until completely dissolved, 15mL is added dropwise and surpasses
Ultrasonic emulsification is carried out using cell crushing instrument simultaneously in pure water, power 300w, time 10min is kept, obtains the breast of stable homogeneous
Liquid；Emulsion is placed in room temperature 32h is stirred under 300rpm, volatilization removes organic solvent and obtains mixing carrier micelle.
Embodiment 4
PAEEP is prepared according to the method for embodiment 112-PLLA70；
Doxorubicin hydrochloride 10.2mg, PEG are weighed respectively83-PLLA70(wherein the weight average molecular weight of PEG block is 5000,
The weight average molecular weight of PLLA blocks is 5000) 80.8mg and PAEEP12-PLLA70(weight average molecular weight of wherein PAEEP blocks is
The weight average molecular weight of 2000, PLLA blocks is 5000) 20.3mg, and the mixing that 1mL chloroforms and 0.5mL methanol are dissolved in jointly is molten
In agent, and 50 μ L triethylamine solutions are added by doxorubicin hydrochloride progress desalination acid treatment, until completely dissolved, 15mL is added dropwise
Ultrasonic emulsification is carried out using cell crushing instrument simultaneously in ultra-pure water, power 300w, time 10min is kept, obtains stable homogeneous
Emulsion；Emulsion is placed in room temperature 24h is stirred under 400rpm, volatilization removes organic solvent and obtains mixing carrier micelle.
Embodiment 5
PAEEP is prepared according to the method for embodiment 121-PLLA90；
Doxorubicin hydrochloride 8.5mg, PEG are weighed respectively100-PLLA90(wherein the weight average molecular weight of PEG block is 8000,
The weight average molecular weight of PLLA blocks is 6400) 60.2mg and PAEEP21-PLLA90(weight average molecular weight of wherein PAEEP blocks is
The weight average molecular weight of 3500, PLLA blocks is 6400) 40.7mg, and the in the mixed solvent of 3mL chloroforms and 1mL methanol is dissolved in jointly,
And 50 μ L triethylamine solutions are added by doxorubicin hydrochloride progress desalination acid treatment, until completely dissolved, 15mL is added dropwise ultrapure
Ultrasonic emulsification is carried out using cell crushing instrument simultaneously in water, power 200w, time 10min is kept, obtains the emulsion of stable homogeneous；
Emulsion is placed in room temperature 24h is stirred under 200rpm, volatilization removes organic solvent and obtains mixing carrier micelle.
Embodiment 6
PAEEP is prepared according to the method for embodiment 121-PLLA120；
Doxorubicin hydrochloride 9.2mg, PEG are weighed respectively100-PLLA90(wherein the weight average molecular weight of PEG block is 8000,
The weight average molecular weight of PLLA blocks is 6400) 72.5mg and PAEEP21-PLLA120(weight average molecular weight of wherein PAEEP blocks is
The weight average molecular weight of 3500, PLLA blocks is 8600) 30.3mg, and the mixing that 1mL dichloromethane and 0.5mL methanol are dissolved in jointly is molten
In agent, and 50 μ L triethylamine solutions are added by doxorubicin hydrochloride progress desalination acid treatment, until completely dissolved, 10mL is added dropwise
Ultrasonic emulsification is carried out using cell crushing instrument simultaneously in ultra-pure water, power 200w, time 10min is kept, obtains stable homogeneous
Emulsion；Emulsion is placed in room temperature 24h is stirred under 200rpm, volatilization removes organic solvent and obtains mixing carrier micelle.
Embodiment 7
PAEEP is prepared according to the method for embodiment 121-PLLA120；
Doxorubicin hydrochloride 8.3mg, PEG are weighed respectively100-PLLA120(wherein the weight average molecular weight of PEG block is 8000,
The weight average molecular weight of PLLA blocks is 8600) 80.8mg and PAEEP21-PLLA120(weight average molecular weight of wherein PAEEP blocks is
The weight average molecular weight of 3500, PLLA blocks is 8600) 20.2mg, and the mixed solvent of 1mL chloroforms and 0.5mL methanol is dissolved in jointly
In, and 50 μ L triethylamine solutions are added by doxorubicin hydrochloride progress desalination acid treatment, until completely dissolved, 10mL is added dropwise and surpasses
Ultrasonic emulsification is carried out using cell crushing instrument simultaneously in pure water, power 200w, time 10min is kept, obtains the breast of stable homogeneous
Liquid；Emulsion is placed in room temperature 24h is stirred under 200rpm, volatilization removes organic solvent and obtains mixing carrier micelle.
Embodiment 8
PAEEP prepared by Example 112-PLLA70, it is 1 according to the amino and RGDF mol ratio on PAEEP side chains:
1.5 weigh respectively corresponding PAEEP-PLLA polymer, RGDF and with RGDF equimolars than 1- ethyls-(3- dimethylaminos
Propyl group) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL), NHS (n-hydroxysuccinimide), co-dissolve in DMSO solvents, its
Middle reactant gross mass is 1 with solvent quality ratio:10, stirring reaction 24h, stir speed (S.S.) 150rpm at room temperature；
Product is positioned over peptide, the EDC that dialysis 24h removings are not coupled in bag filter (MWCO=1000) by reaction after terminating
And the small molecular weight impurity such as NHS, white RGDF-PAEEP-PLLA powder is obtained after vacuum freeze drying 48h.
The PAEEP prepared respectively to embodiment 1 using nuclear magnetic resonance12-PLLA70The RGDF- prepared with embodiment 8
PAEEP-PLLA is detected that acquired results are as shown in Figure 1；
The PAEEP prepared respectively to embodiment 1 using infrared spectrometer12-PLLA70The RGDF- prepared with embodiment 8
PAEEP-PLLA is detected that acquired results are as shown in Figure 2；
Can be seen that the products therefrom of embodiment 8 according to Fig. 1 and Fig. 2 is RGDF-PAEEP-PLLA really, and RGDF is successfully repaiied
Decorations are in PAEEP12-PLLA70PAEEP blocks on, and RGDF block molecules amount be 8k.
Embodiment 9
Method according to embodiment 8 prepare RGDF-PAEEP-PLLA (wherein the weight average molecular weight of PAEEP blocks be 2000,
Block quantity is 12；The weight average molecular weight of PLLA blocks be 5000, block quantity for 70, RGDF quality account for PEG-PLLA and
RGDF-PAEEP-PLLA gross masses 6.5%)；
Weigh 9.7mg doxorubicin hydrochlorides, 87.5mg PEG-PLLA (wherein the weight average molecular weight of PEG block is 5000,
5000) and 12.5mg RGDF-PAEEP-PLLA the weight average molecular weight of PLLA blocks is, is dissolved in 1mL dichloromethane and 1mL jointly
The in the mixed solvent of ethanol, and 100 μ L triethylamine solutions are added by doxorubicin hydrochloride progress desalination acid treatment, wait to be completely dissolved
Afterwards, it is added dropwise in 20mL ultra-pure waters and carries out ultrasonic emulsification using cell crushing instrument simultaneously, keeps power 200w, time 5min,
The emulsion of stable homogeneous is obtained, emulsion is placed in room temperature 24h is stirred under 200rpm, volatilization removing organic solvent obtains RGDF and repaiied
The carrier micelle of decorations, is designated as RGDF-M (6.5%).
Embodiment 10
Method according to embodiment 8 prepare RGDF-PAEEP-PLLA (wherein the weight average molecular weight of PAEEP blocks be 2000,
Block quantity is 12；The weight average molecular weight of PLLA blocks be 5000, block quantity for 70, RGDF quality account for PEG-PLLA and
RGDF-PAEEP-PLLA gross masses 8.5%)；
Weigh 10.5mg doxorubicin hydrochlorides, 80.5mg PEG-PLLA (wherein the weight average molecular weight of PEG block is 5000,
5000) and 20.0mg RGDF-PAEEP-PLLA the weight average molecular weight of PLLA blocks is, is dissolved in 3mL chloroforms and 1mL ethanol jointly
In the mixed solvent, and add 100 μ L triethylamine solutions by doxorubicin hydrochloride carry out desalination acid treatment, until completely dissolved, by
It is added dropwise in 15mL ultra-pure waters while carrying out ultrasonic emulsification using cell crushing instrument, holding power 200w, time 5min are obtained
One stable emulsion, emulsion is placed in room temperature 24h is stirred under 200rpm, and volatilization removes the load that organic solvent obtains RGDF modifications
Medicine micella, is designated as RGDF-M (8.5%).
Embodiment 11
Method according to embodiment 8 prepare RGDF-PAEEP-PLLA (wherein the weight average molecular weight of PAEEP blocks be 2000,
Block quantity is 12；The weight average molecular weight of PLLA blocks be 5000, block quantity for 70, RGDF quality account for PEG-PLLA and
RGDF-PAEEP-PLLA gross masses 10.5%)；
Weigh 10.5mg doxorubicin hydrochlorides, 80.5mg PEG-PLLA (wherein the weight average molecular weight of PEG block is 5000,
5000) and 20.0mg RGDF-PAEEP-PLLA the weight average molecular weight of PLLA blocks is, is dissolved in 3mL chloroforms and 1mL ethanol jointly
In the mixed solvent, and add 100 μ L triethylamine solutions by doxorubicin hydrochloride carry out desalination acid treatment, until completely dissolved, by
It is added dropwise in 15mL ultra-pure waters while carrying out ultrasonic emulsification using cell crushing instrument, holding power 200w, time 5min are obtained
One stable emulsion, emulsion is placed in room temperature 24h is stirred under 200rpm, and volatilization removes the load that organic solvent obtains RGDF modifications
Medicine micella, is designated as RGDF-M (10.5%).
Embodiment 12
Method according to embodiment 8 prepare RGDF-PAEEP-PLLA (wherein the weight average molecular weight of PAEEP blocks be 2000,
Block quantity is 12；The weight average molecular weight of PLLA blocks be 5000, block quantity for 70, RGDF quality account for PEG-PLLA and
RGDF-PAEEP-PLLA gross masses 5.2%)；
Weigh 10.5mg doxorubicin hydrochlorides, 80.5mg PEG-PLLA (wherein the weight average molecular weight of PEG block is 5000,
5000) and 20.0mg RGDF-PAEEP-PLLA the weight average molecular weight of PLLA blocks is, is dissolved in 3mL chloroforms and 1mL ethanol jointly
In the mixed solvent, and add 100 μ L triethylamine solutions by doxorubicin hydrochloride carry out desalination acid treatment, until completely dissolved, by
It is added dropwise in 15mL ultra-pure waters while carrying out ultrasonic emulsification using cell crushing instrument, holding power 200w, time 5min are obtained
One stable emulsion, emulsion is placed in room temperature 24h is stirred under 200rpm, and volatilization removes the load that organic solvent obtains RGDF modifications
Medicine micella, is designated as RGDF-M (5.2%).
Embodiment 13
(1) particle diameter distribution of the PEG-PLLA/PAEEP-PLLA mixing carrier micelles prepared to embodiment 2~7 is divided
Analysis, acquired results are as shown in table 1：
The particle diameter distribution of PEG-PLLA/PAEEP-PLLA mixing carrier micelles prepared by the embodiment 2~7 of table 1
Data in table 1 can be seen that：PEG-PLLA/PAEEP-PLLA mixing carrier micelle particle diameter distributions are located at
Between 160~190nm, and PDI is smaller, represents that particle diameter distribution is homogeneous.
(2) micella study on the stability：By embodiment 2~7 prepare PEG-PLLA/PAEEP-PLLA mixing carrier micelle and
24h is incubated altogether under the conditions of 37 DEG C after PBS (pH 7.4,10mM) mixing containing BSA (concentration is 0.25mg/mL), so
After detect its particle diameter distribution change, the results are shown in Table 2.
Particle diameter distribution and Zeta potential of the PEG-PLLA/PAEEP-PLLA mixing carrier micelle of table 2 after 24h is incubated
According to table 2 as can be seen that change of size is little after PEG-PLLA/PAEEP-PLLA mixing carrier micelle incubation 24h,
Show that mixing carrier micelle prepared by the present invention has good stability.
(3) particle diameter distribution to RGDF prepared by embodiment 9~12 mixing carrier micelle modified is detected, gained knot
Fruit is as shown in table 3.
The particle diameter distribution of the mixing carrier micelle of the RGDF of table 3 modifications
The mixing carrier micelle particle diameter distribution that data in table 3 can be seen that RGDF modifications is located at 160~190nm
Between, and PDI is smaller, represents that particle diameter distribution is homogeneous.
(4) carrier micelle prepared to the embodiment of the present invention 2 (being designated as Control-M), 9 (RGDF-M (6.5%)) is not
With vitro drug release studies are carried out under pH value condition, while being carried out to PEG-PLLA carrier micelles common in this area external
Drug release studies, are as a result shown in Fig. 3.
As a result show, in the environment of pH 7.4, the release behavior of three groups of micellas is basically identical, and burst size is basic after 24h
Reach saturation.During pH 6.5, RGDF-M (6.5%) micella rate of release is substantially accelerated, and in pH 4.5, PEG-PLLA micellas
Burst size has reached that 75%~80% or so, Control-M and the burst size of RGDF-M (6.5%) micella are then basically reached
100%.It follows that the mixing carrier micelle of the mixing carrier micelle and RGDF modifications after addition PAEEP-PLLA is in low pH
Under conditions of, faster, this depends on the PAEEP-PLLA quick characteristic of acid-sensitive and enzyme to drug release rate.
(5) flow cytomery Bel7402 cells are used to embodiment 2 under conditions of pH7.4 and pH6.5 respectively
Prepared by (being designated as Control-M), 9 (RGDF-M (6.5%)), 10 (RGDF-M (8.5%)), 11 (RGDF-M (10.5%)) mixed
The endocytosis amount of carrier micelle and the common PEG-PLLA carrier micelles in this area is closed, result of the test is shown in Fig. 4, Fig. 5 and Fig. 6 institute
Show.
Bel7402 cells are can be seen that according to Fig. 4 result to become the endocytosis amount of PEG-PLLA micellas without pH sensitiveness
Change；Bel7402 cells are also little for Control-M endocytosis amount sensitiveness, but interior under conditions of pH7.4 and pH6.5
The amount of gulping down is all apparently higher than PEG-PLLA micellas；Endocytosis amounts of the RGDF-M (6.5%) under conditions of pH 7.4 is significantly less than pH6.5
Under the conditions of endocytosis amount, illustrate in neutral conditions, RGDF modification mixing carrier micelle in RGDF be hidden in PEG hydrated sheaths
In, and under pH 6.5 solutions of weak acidity, the protonated amino degree increase that RGDF is carried in itself and on PAEEP side chain,
Add the extension degree of PAEEP blocks so that RGDF realizes Targeting Effect exposed to micellar surface, therefore its endocytosis amount with
PEG-PLLA groups are compared and dramatically increased.
According to Fig. 5 as can be seen that Bel7402 cells are to RGDF-M (8.5%) glue under conditions of pH 7.4 and pH 6.5
The endocytosis of beam is apparently higher than PEG-PLLA micellas.
According to Fig. 6 as can be seen that in (10.5%) one group of RGDF-M, in pH 7.4, because RGDF contents are higher, because
And endocytosis of the target cell to it can be dramatically increased under conditions of pH 7.4 and pH 6.5.
(6) quantitatively detect that mouse monokaryon macrophage RAW264.7 (is designated as to the embodiment of the present invention 2 using flow cytometer
Control-M), the endocytosis amount of 9 and 11 carrier micelles prepared, mouse monokaryon macrophage RAW264.7 and full culture medium are existed
2h is cultivated under the conditions of 37 DEG C, wherein including Ah mould of desalination hydrochlorate in 10%FBS and carrier micelle, carrier micelle in full culture medium
Plain (Dox) concentration is 5 μ g/mL；After 2h, PBS vitellophag is used, with flow cytomery cell to carrying after being collected by centrifugation
The endocytosis amount of medicine micella, testing result is as shown in Figure 7.
Can be had according to Fig. 7 result find out with PEG-PLLA micellar phases ratio, the cells of RAW 264.7 to Control-M,
The endocytosis amount of RGDF-M (10.5%) and RGDF-M (6.5%) mixing carrier micelles is declined slightly, because Control-M,
Possess PEG/PAEEP mixing hydrophilic layer in RGDF-M (10.5%) and RGDF-M (6.5%) mixing carrier micelles, therefore have
Even more excellent anti-protein adsorption ability similar with PEG-PLLA micellas, this shows that mixing carrier micelle has anti-monokaryon macrophage thin
The ability of born of the same parents' system phagocytosis, thus possess higher passive target efficiency, the treatment for tumour is most important.
(7) prepared by embodiment 2 (being designated as Control-M), 9 (RGDF-M (6.5%)), 11 (RGDF-M (10.5%))
It is clear that medicine, PBS are discarded after the common incubation 12h of carrier micelle, common PEF-PLLA carrier micelles and Bel7402 cells, 12h
Wash, cell is fixed, DAPI is dyed and mounting, the situation of insoluble drug release, observation result such as Fig. 8 are observed with confocal microscope
It is shown；
According to Fig. 8 as can be seen that Dox fluorescence is very weak in the nucleus of PEG-PLLA micella groups, this shows that intracellular discharges
Adriamycin amount seldom, and mixing adriamycin amount in the nucleus of carrier micelle group substantially increases, and RGDF-M (10.5%) and
Doxorubicin fluorescence intensity is stronger compared to Control-M in the core that (6.5%) two group of RGDF-M, and this shows be mixed with PAEEP-
PLLA mixing carrier micelle is dramatically increased in intracellular drug release rate, and this depends on PAEEP acid-sensitive and enzyme sensitiveness,
And it has been coupled the micella intracellular rate of release of RGDF targeting peptides faster.
(8) from 12 week old normal balb/c female mices, respectively be injected intravenously embodiment 2 (being designated as Control-M),
Carrier micelle, common PEG-PLLA carrier micelles and trip prepared by 9 (RGDF-M (6.5%)), 11 (RGDF-M (6.5%))
From adriamycin, control injection doxorubicin dosages all be 5mg/kg, take blood from the orbital venous plexus of mouse in different time points
Obtain after serum, using volume ratio as 5:1 ratio methanol dilution serum, Dox in blood is calculated using the method for fluorescent quantitation
Content, set up Drug-time curve, acquired results are as shown in Figure 9；
According to Fig. 9 as can be seen that after free Doxorubicin intravenous injections 1h, the medicament contg contained in blood declines rapidly；
And PEG-PLLA micellas, RGDF-M (10.5%), Control-M and the corresponding concentration difference of RGDF-M (6.5%) experimental group
For the 12.3 of adriamycin group concentration of dissociating, 12.7,16.3 and 18.7 times.This shows to possess PAEEP/PEG hydrophobes microfacies point
There is the anti-protein adsorption ability more excellent than simple PEG-PLLA micellas from the mixing carrier micelle at interface, micella is added in body
The interior long circulating time.And RGDF-M (6.5%) is compared with RGDF-M (10.5%), blood concentration after 1h the former be the latter
1.5 times, this shows that RGDF-M (6.5%) possesses stronger anti-protein adsorption ability and long circulating ability.
(9) in order to more intuitively observe distribution situation of the mixing carrier micelle in Mice Body, the present invention is respectively using trip
From adriamycin, embodiment 2 (being designated as Control-M), 9 (RGDF-M (6.5%)), 11 (RGDF-M (10.5%)) prepare load
Medicine micella and PEG-PLLA carrier micelles parcel fluorescent dye DIR, are then injected intravenously to Mice Body, aobvious with near-infrared image
Show the internal distribution situation of mixing carrier micelle, acquired results are as shown in Figure 10；
According to Figure 10 as can be seen that compared with free adriamycin group, other four groups of micellas have obvious tumor locus
Accumulation, the cumulant highest of tumor locus after 48h, and the accumulation of the tumor locus of RGDF-M (6.5%) groups is most；
The fluorescence intensity of RGDF-M (10.5%) micella group tumor locus is higher than PEG-PLLA micellas group but less than Control-M
Group, this is due to the more RGDF modifications in surface simultaneously and adds endocytosis of the mononuclear phagocyte system to it.Free Ah mould
The fluorescence of element group is distributed mainly on liver and spleen, and the tumour fluorescence intensity of RGDF-M (6.5%) groups is most strong, but in liver and
Spleen fluorescence intensity is weaker, and this has absolutely proved that its anti-mononuclear phagocyte system (MPS) phagocytic activity is most strong.
(10) in order to further study internal distribution situation of the carrier micelle in mouse, the present invention has investigated the (note of embodiment 2
For Control-M), 9 (RGDF-M (6.5%)), 11 (RGDF-M (10.5%)) prepare carrier micelle carrier micelle through vein
24h distribution situation after being injected into Mice Body, acquired results are as shown in figure 11；
According to Figure 11 as can be seen that the doxorubicin content of tumor locus, RGDF-M (6.5%) micella groups are apparently higher than free
Adriamycin, PEG-PLLA micellas and RGDF-M (10.5%) groups (p<0.05)；And mixing carrier micelle group Control-M,
RGDF-M (10.5%) and RGDF-M (6.5%) groups are significantly lower than PEG-PLLA group micellas in the cumulant of liver and spleen
(p<0.01), experimental result further demonstrates the superiority of mixing carrier micelle.
(11) using free adriamycin, embodiment 2 (being designated as Control-M), 9 (RGDF-M (6.5%)), 11 (RGDF-M
(10.5%)) carrier micelle and PEG-PLLA carrier micelles prepared is treated to tumor-bearing mice, and treatment cycle is 21 days, system
Gross tumor volume and mouse weight change in therapeutic process are counted, as a result as shown in Figure 12~Figure 13；
Tumor weight is detected after to treating 21 days, as a result as shown in figure 14；
The tumour after treating 21 days is observed using x-ray spectrometer, observation result is as shown in figure 15；
It is can be seen that according to Figure 12~Figure 15 result after treatment 21 days, no matter in gross tumor volume or in tumour weight
In amount, the tumor killing effect of three groups of mixing carrier micelle systems is then substantially better than PEG-PLLA micellas group and free adriamycin group；And
And the antitumous effect that RGDF-M (6.5%) is organized in mixing carrier micelle is preferably, the mixing carrier micelle of this explanation RGDF modifications
Middle RGDF content can influence therapeutic effect.
As seen from the above embodiment, the invention provides the mixing carrier micelle based on polyphosphate have it is good steady
It is qualitative, have internal long circulating effect concurrently and promote the effect of target cell endocytosis, and easily carry out targeting modification；The master that the present invention is provided
RGDF of the moving-target into the mixing carrier micelle of base group modification can be able to hidden under conditions of pH 7.4 in PEG hydrated sheath
Hide, it is to avoid the phagocytosis of normal structure, reach after tumor locus, the slightly sour environment of tumor locus enables RGDF to expose, and reaches master
Moving-target to effect, further increase target cell endocytosis so that further improve tumour therapeutic effect；What the present invention was provided
The oncotherapy excellent effect of the mixing carrier micelle of mixing carrier micelle and active targeting base group modification based on phosphate, it is bright
It is aobvious to be better than single PEG-PLLA carrier micelles.
As seen from the above embodiment, it is only the preferred embodiment of the present invention that the present invention is described above, it is noted that for
For those skilled in the art, under the premise without departing from the principles of the invention, can also make it is some improvement and
Retouching, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of mixing carrier micelle based on polyphosphate, including carrier and the hydrophobicity chemotherapeutic that is coated by the carrier
Thing；The carrier includes polyphosphate-PLA di-block copolymer and PEG2000-PLA12500；
The polyphosphate-PLA di-block copolymer has structure shown in Formulas I：
In Formulas I, m=5~30, n=10~150.
2. mixing carrier micelle according to claim 1, it is characterised in that the block of polyethylene glycol-polylactic acid two is total to
The mass ratio of polymers and polyphosphate-PLA di-block copolymer is (1~20):(1~5).
3. mixing carrier micelle according to claim 1 or 2, it is characterised in that the block of polyphosphate-PLA two
The weight average molecular weight of PLA block is 2000~20000 in copolymer；The weight average molecular weight of polyphosphate block be 2000~
10000；
The weight average molecular weight of PLA block is 2000~20000 in the PEG2000-PLA12500；Poly- second
The weight average molecular weight of diol block is 2000~10000.
4. mixing carrier micelle according to claim 1, it is characterised in that the matter of the hydrophobicity chemotherapeutics and carrier
Amount is than being (0.1~2):10.
5. mixing the preparation method of carrier micelle described in Claims 1 to 4 any one, comprise the following steps：
By polyphosphate-PLA di-block copolymer, PEG2000-PLA12500, hydrophobicity chemotherapeutics,
Organic basic compound and organic solvent mixing, obtain oil phase；
Oil phase is added to the water and emulsified, emulsion is obtained；
Organic solvent in emulsion is removed, the mixing carrier micelle based on polyphosphate is obtained.
6. preparation method according to claim 5, it is characterised in that the organic basic compound includes triethylamine, second
One or more in diamines and pyridine.
7. the preparation method according to claim 5 or 6, it is characterised in that the organic basic compound and hydrophobicity
The mol ratio for treating medicine is (1~10):1.
8. preparation method according to claim 5, it is characterised in that the organic solvent is the mixed of chloralkane and alcohols
Bonding solvent.
9. a kind of active targeting base group modification mixing carrier micelle, including the mixing load medicine described in Claims 1 to 4 any one
Mixing carrier micelle and modify in mixing load medicine glue prepared by preparation method described in micella or claim 5~8 any one
Active targeting group on Shu Zaiti；
The active targeting group is coupled on the polyphosphate block of polyphosphate-PLA di-block copolymer；
The active targeting group is arginine-glycine-aspartic acid-phenylalanine peptide.
10. active targeting base group modification mixing carrier micelle according to claim 9, it is characterised in that the active target
It is that polyphosphate-PLA di-block copolymer in mixing carrier micelle and polyethylene glycol-polylactic acid two are embedding to the quality of group
The 1%~20% of section copolymer gross mass.
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| CN108042490A (en) * | 2018-01-10 | 2018-05-18 | 华中科技大学 | Nano medicament carrying system, its preparation method, pharmaceutical composition and the application in treating cancer |
| CN110801432A (en) * | 2019-11-12 | 2020-02-18 | 重庆医科大学 | Multifunctional amphiphilic camptothecin-fluorouracil-loaded targeting mixed micelle drug delivery system and preparation method thereof |
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